Molecular methods of hybrid seed production

ABSTRACT

A process is described for producing fertile hybrid seed or hybrid seed comprising fertile and sterile seed using male-sterile plants created by employing molecular techniques to manipulate anti-sense gene and other genes that are capable of controlling the production of fertile pollen in plants. Said plants are functionally male-sterile plants with pollen from male-fertile plants. Hybrid seed production is simplified and improved by this invention and can be extended to plant crop species for which commercially acceptable hybrid seed production methods are not currently available.

This application is a divisional of application Ser. No. 08/897,325filed Jul. 21, 1997, now Abandoned, which is a divisional of applicationSer. No. 08/276,510 filed Jul. 14, 1994, which is a continuation ofapplication Ser. No. 07/556,917 filed Jul. 20, 1990, now Abandoned,which is a 371 of PCT/CA90/00037 filed Feb. 2, 1990, which is acontinuation-in-part of application Ser. No. 07/306,438 filed Feb. 3,1989, now Abandoned, which is a continuation-in-part of application Ser.No. 07/151,906 filed Feb. 3, 1988, now Abandoned.

TECHNICAL FIELD

The present invention relates to a method for producing male sterileplants and hybrid seed, to genetic material used to impart the malesterility trait and to new products produced by said method, namely,genetically transformed plants carrying the male sterile trait, malesterile plants and hybrid seed produced by pollinating said plants withpollen from male fertile plants.

BACKGROUND ART

Production of hybrid seed for commercial sale is a large industry.Hybrid plants grown from hybrid seed benefit from the heterotic effectsof crossing two genetically distinct breeding lines. The agronomicperformance of this offspring is superior to both parents, typically invigour, yield, and uniformity. The better performance of hybrid seedvarieties compared to open-pollinated varieties makes the hybrid seedmore attractive for farmers to plant and thereby commands a premiumprice in the market place.

In order to produce hybrid seed uncontaminated with selfed seedpollination control methods must be implemented to ensurecross-pollination and not self-pollination. Pollination controlmechanisms can be mechanical, chemical, or genetic.

A simple mechanical method for hybrid seed production can be used if theplant species in question has spat-ally separate male and female flowersor separate male and female plants. The corn plant, for example, haspollen producing male flowers in an inflorescence at the apex of theplant and female flowers in the axils of leaves along the stem.Outcrossing is assured by mechanically de-tasselling female plants toprevent selfing.

Most major crop plants of interest, however, have both functional maleand female organs within the same flower so emasculation is not a simpleprocedure. It is possible to remove by hand the pollen forming organsbefore pollen shed, however this form of hybrid seed production isextremely labour intensive and hence expensive. Seed is produced in thismanner if the value and amount of seed recovered warrants the effort.

A second general method of producing hybrid seed is to use chemicalsthat kill or block viable pollen formation. These chemicals, termedgametocides, are used to impart a transitory male-sterility. Commercialproduction of hybrid seed by use of gametocides is limited by theexpense and availability of the chemicals and the reliability and lengthof action of the applications. These chemicals are not effective Forcrops with an extended flowering period because new flowers will beproduced that will not be affected. Repeated application of chemicals isimpractical because of costs.

Many current commercial hybrid seed production systems for field cropsrely on a genetic method of pollination control. Plants that are used asfemales either fail to make pollen, fail to shed pollen or producepollen that is biochemically unable to effect self-fertilization. Plantsthat are unable (by several different means) to self pollinatebiochemically are termed self-incompatible. Difficulties associated withthe use of self-incompatibilities are: availability and propagation ofthe self-incompatible female line and stability of theself-incompatibility. In some instances self-incompatibility can beovercome chemically or immature buds can be pollinated by hand beforethe biochemical mechanism that blocks pollen is activated.Self-incompatible systems that can be deactivated are often veryvulnerable to stressful climatic conditions that break or reduce theeffectiveness of the biochemical block to self-pollination.

Of more widespread interest for commercial seed production are systemsof pollen control based on genetic mechanisms causing male sterility.These systems are of two general types: (a) genic male sterility, whichis the failure of pollen formation because of one or more nuclear genesor (b) cytoplasmic-genetic male sterility (commonly called cytoplasmicmale sterility or CMS) in which pollen formation is blocked or abortedbecause of a defect in a cytoplasmic organelle (mitochondrion) (forgeneral discussions on genic sterility, CMS and hybrid formation inplants see Frankel, R., et al., Pollination Mechanisms, Reproduction andPlant Breeding; Springer V., et al., Monographs on Theoretical andApplied Genetics, N.Y, 1977; Edwardson, J. P., Bot. Rev. 36:341-420,1970).

Nuclear (genic) sterility can be either dominant or recessive. Adominant sterility can only be used for hybrid seed production iffertility of the hybrid plants is not critical and if propagation of thefemale line is feasible (eg. by clonal propagation or by the use of aselectable marker closely linked to the sterility gene).

Many successful hybridization schemes involve the use of CMS. In thesesystems, a specific mutation in the cytoplasmically locatedmitochondrion can, when in the proper nuclear background, lead to thefailure of mature pollen formation. In some other instances, the nuclearbackground can compensate for the cytoplasmic mutation and normal pollenformation occurs. The nuclear trait that allows pollen formation inplants with CMS mitochondria is called restoration and is the propertyof specific "restorer genes". Generally the use of CMS for commercialseed production involves the use of three breeding lines, themale-sterile line (female parent), a maintainer line which is isogenicto the male-sterile line but contains fully functional mitochondria andthe male parent line.

The male parent line may carry the specific restorer genes (usuallydesignated a restorer line) which then imparts fertility to the hybridseed. For crops (eg. vegetables) for which seed recovery from the hybridis unimportant, a CMS system could be used without restoration. Forcrops for which the fruit or seed of the hybrid is the commercialproduct then the fertility of the hybrid seed must be restored byspecific restorer genes in the male parent or the male-sterile hybridmust be pollinated. Pollination of non-restored hybrids can be achievedby including with hybrids a small percentage of male fertile plants toeffect pollination. In most species, the CMS trait is inheritedmaternally (because all cytoplasmic organelles are inherited from theegg cell only), which can restrict the use of the system.

In a crop of particular interest herein, the oilseed crop of the speciesBrassica napus or Brassica campestris, commonly referred to as Canola,no commercial hybrid system has been perfected to date. Mechanicalemasculation of flowers is not practical for hybrid seed production onany scale. The use of currently available gametocides is impracticalbecause of the indeterminate nature of flower production. Repeatedapplication of chemicals is expensive and the method is prone tocontamination with selfed seed.

Genes that result in self-incompatibility are quite widespread inBrassica species and self-incompatible hybrid systems have been used forhybrid seed production in vegetables. Major difficulties are associatedwith the propagation of the female lines and the breakdown ofself-incompatibilities under stressful conditions. Adaptation of thesesystems to Brassica oilseeds is restricted by the expense of increasingthe female lines and the availability of appropriate self-incompatiblegenes in the dominant Canola species, Brassica napus.

A variety of sources of male sterility are available in Brassicaspecies. Both recessive and dominant genic systems have been reported,however their use is restricted because large scale in vitro propagationor roguing of female lines is in most cases impractical for large scaleseed production.

Additionally, a number of CMS systems have been reported in Brassicaspecies. Four of these systems have been explored as possible vehiclesfor hybrid seed production: pol, nap, anand and ogu. The Polima system(pol) has been widely studied and is probably the closest to commercialuse. Good restoration and maintenance of pol CMS has been achieved,however the system suffers from potential instability of the CMS withhigh temperature, a reduction in the heterotic effect of crossingdifferent lines (because of the defective mitochondria) and a reductionin hybrid seed oil content. The use of other CMS systems is alsorestricted by heat sensitivity (nap), difficulty in restoration offertility (ogu, anand), difficulty in the maintenance of the sterility(nap) and low temperature chlorosis associated with the sterilecytoplasm (ogu). Improvement of these systems is the object ofconsiderable research, however all of the systems have some inherentweaknesses that limit their utility.

For a general discussion of male sterility in Brassica see Shiga, T.,Male Sterility and Cytoplasmic Differentiation. In Brassica Crops andWild Alles, Biology and Breeding, Japan Scientific Societies Press,Tokyo pp. 205-221; Thompson, K. F., Heredity 29:253-257).

It is recognized that a desirable system for hybrid seed production inany crop would be a form of genic male sterility that could be regulatedor overcome to allow male fertility for the propagation or increase ofthe female lines or to allow fertility in hybrids. This recognition hasstimulated research on the use of molecular systems to effect genic malesterility that could be used for hybrid seed formation. In addition, theadvent and widespread application of recombinant DNA techniques mayprovide a mechanism of introduction of novel DNA sequences into a widevariety of different crop species that is not possible by the limitedsexual methods of genetic exchange between different species. Amolecular approach has the advantage that the hybridization system canbe imposed on all breeding lines or cultivars of any given crop withoutthe need for extensive backcrossing and disruption of established inbredlines leading to the rapid production of male sterile lines with wellcharacterized and superior agronomic performance.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant DNA molecule for use inthe preparation of a male-sterile plant or a plant carrying amale-sterile trait and reproductive material of said plants, comprising(a) one or more DNA sequences which may be the same or different, whichencode a gene product which when produced in a cell of a plant which isessential to pollen formation and/or function is directly or indirectlycapable of substantially interfering with the function and/ordevelopment of said cell; and (b) one or more promoters which may be thesame or different, said promoters being capable of regulating theexpression of said DNA sequences; and wherein the DNA sequences andpromoters are selected such that the gene product selectively interfereswith the function and/or development of a cell of a plant that isessential to pollen formation and/or function and whereby a plantregenerated from a cell of a plant having said recombinant DNA moleculeintegrated into its genome is substantially male-sterile or carries themale sterile trait.

A gene product which is directly capable of interfering with thefunction and/or development of a cell of a plant which is essential topollen formation and/or function includes a protein or polypeptide whichis substantially cytotoxic to the cell, or a nucleotide sequence whichinterferes with the expression of a gene which is essential to pollenformation and/or function or a gene which is essential to the continueddevelopment and/or function of all metabolically competent cells of aplant. A gene product which is indirectly capable of interfering withthe function and/or development of a cell of a plant which is essentialto pollen formation and/or function includes a protein or polypeptidewhich renders the cell susceptible to a chemical agent or physiologicalstress or a protein or polypeptide which render a non-toxic substancesubstantially cytotoxic to the cell.

In one embodiment of the invention a recombinant DNA molecule isprovided which comprises at least one DNA sequence which encodes a geneproduct which when produced in a cell of a plant which is essential topollen formation and/or function is substantially cytotoxic to saidcell.

In a preferred embodiment of the invention a recombinant DNA molecule isprovided which comprises at least one DNA sequence which is ananti-sense gene which encodes an RNA which substantially interferes withthe expression of a sense gene which is essential to pollen formationand/or function of a plant.

In another preferred embodiment of the invention a recombinant DNAmolecule is provided which comprises at least one DNA sequence which isan anti-sense gene which encodes an RNA which substantially interfereswith the expression of a sense gene which is essential to the continueddevelopment and/or function of all metabolically competent cells of aplant.

In a further preferred embodiment of the invention a recombinant DNAmolecule is provided comprising at least one DNA sequence which encodesa protein or polypeptide which when produced in a cell of a plant whichis essential to pollen formation and/or function is substantiallycytotoxic to said cell.

In another embodiment of the invention a recombinant DNA molecule isprovided comprising at least one DNA sequence which encodes a geneproduct which when produced in a cell of a plant which is essential topollen formation and/or function renders a non-toxic substance cytotoxicto said cell. The recombinant DNA molecule may additionally comprise asecond DNA sequence which encodes a second gene product which converts asubstance which is endogenous to a plant cell to the non-toxicsubstance.

In still another embodiment of the invention a recombinant DNA moleculeis provided comprising at least one DNA sequence which is an anti-sensegene which encodes an RNA which substantially interferes with theexpression of a sense gene which confers on cells of a plant resistanceto a chemical agent or physiological stress.

The recombinant DNA molecules of the invention comprise a promoter(s)which promoter(s) may be selected from the group of promoters consistingof a constitutive promoter, an inducible promoter which is activethroughout pollen formation or during Transcription of one or more ofthe DNA sequences; and, a pollen specific promoter which, regulates theexpression of one or more of the DNA sequences selectively in cells of aplant which are essential to pollen formation and/or function.

The invention also relates to a pollen specific promoter which regulatesthe expression of a DNA sequence selectively in cells of a plant whichare essential to pollen formation and/or function.

The present invention additionally relates to a plasmid vectorcontaining one or more recombinant DNA molecules of the invention whichvector is adapted to transform plant cells, a plant cell containing arecombinant DNA molecule of the invention, a plant cell culturecontaining the plant cell, a plant containing the plant cell and a seedof such a plant, a process for producing a male-sterile plant, a processof producing a hybrid seed and hybrid seed so produced, and a hybridplant obtained by growing the hybrid seed and a hybrid seed producedfrom the hybrid plant.

The following advantages over other hybridization systems may beobtained using the methods according to the present invention:

(a) Hybrid seed production is not labour intensive and can be achievedon a large scale with commercially acceptable costs;

(b) Male sterility is simply inherited and stable in response toenvironmental stresses that limit the effectiveness ofself-incompatibility and CMS based schemes;

(c) Seed that is produced will be relatively uncontaminated by selfedseed;

(d) The system avoids the use of defective cytoplasmic organelles thatmay detract from the performance of hybrid seed;

(e) The system will greatly speed the development and increase thenumber of lines that can be tested as parents in a hybrid cross becauseit can be imposed on any plant or. inbred line capable of beingtransformed and regenerated into plants without the inclusion ofadditional genomic DNA. Additionally plant lines can be tested forcombining ability before inclusion of the hybridizing system which canmodify breeding strategy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the construct-on of an anti-sense genevector that was used for anti-sense RNA inhibition of Beta-glucuronidasegene activity in transgenic plants.

FIG. 2a is a schematic representation of a restriction map and codingregion of clone number L 4, a microspore specific clone isolated from aBrassica napus genomic library. The clone contains three similar copiesof a single gene. These genes are identified as Bp4A, 3p4B and Bp4C. Thefirst (Bp4A) and third (Bp4C) genes are functional, the second gene hasmodifications chat most likely render it non-functional. The restrictionmap is made diagrammatically in that the non-transcribed regions areshown as a single line, while the transcribed regions are shown as aboxed area. The second gene (Bp4C) is identified on the basis ofsequence homology and i-s shown as a boxed area with a dotted line. Thenotation "del 220" refers to an approximately 220 base pairdeletion/rearrangement that has presumably inactivated the second gene(Bp4B) in this clone. Start of transcription is located at the leftmostside of each boxed area (except in the case of gene Bp4B) and exon andintron positions are noted by the exons being filled in with black andthe intron positions being left unfilled. A small arrowhead is shown onthe non-transcribed 5' region of each gene, this arrowhead serves toindicate the promoter region of each gene. Restriction sites areIdentified such that the number of the first nucleotide of therestriction enzyme recognition site is shown. Not all restriction sitesare shown, only those relative to the constricts detailed within areindicated. The genes are presented with the 5' region on the left sideand the 3' region on the right side. The numeration of the DNA sequenceis from left to right, 5' to 3' in all cases.

FIG. 2b is a schematic representation of a restriction map and codingregion of clone number L 10, a microspore specific clone isolated from aBrassica napus genomic library. The clone contains a single gene. Startof transcription, exon, intron and promoter positions are noted as inFIG. 2a. Restriction sites are identified such that the number of thefirst nucleotide of the restriction enzyme recognition site is shown.The genes are presented with the 5' region on the left side and the 3'region on the right side. The numeration of the DNA sequence is fromleft to right, 5' to 3' in all cases.

FIG. 2c is a schematic representation of the restriction map and codingregion of clone number L 16, a microspore specific clone isolated from aBrassica napus genomic library. The clone contains a single gene thatshows similarity to clone L 10. Intron and exon positions are noted asin FIG. 1. Restriction sites are identified such that the number of thefirst nucleotide of the restriction enzyme recognition site is shown.The genes are presented with the 5' region on the left side and the 3'region on the right side. The numeration of the DNA sequence is fromleft to right, 5' to 3' in all cases.

FIG. 2d is a schematic representation of the restriction map and codingregion of clone number L 19, a microspore specific clone isolated from aBrassica napus genomic library. The clone contains a single gene. Startof transcription, exon, intron and promoter positions are noted as inFIG. 1. Restriction sites are identified such that the number of thefirst nucleotide of the restriction enzyme recognition site is shown.The genes are presented with the 5' region on the left side and the 3'region on the right side. The numeration of the DNA sequence is fromleft to right, 5' to 3' in all cases.

FIG. 3a is the complete nucleotide sequence of the clone L 4 representedin FIG. 2a. Only the coding strand is shown for clarity.

FIG. 3b is the nucleotide sequence of the portion of the clone L 10shown in Figure as being underlined in FIG. 2b. Only the coding strandis shown for clarity.

FIG. 3c is the nucleotide sequence of the portion of the clone L 16shown in Figure as being underlined in FIG. 2c. Only the coding strandis shown for clarity.

FIGS. 3d-3y is the nucleotide sequence of the portion of the clone L 19shown in Figure as being underlined in FIG. 2d. Only the coding strandis shown for clarity.

FIG. 4 is the nucleotide sequence of 3 cDNA clones isolated from amicrospore derived cDNA library of Brassica napus. These clones arenamed cBp401, cBp405, and cBp408. These three cDNA clones are extremelyhomologous to members of the L4 Brassica napus microspore specific genefamily (Bp4A, Bp4B, Bp4C). The nucleotide sequence of two of these 3members of the L4 Brassica napus microspore specific gene family areshown in this Figure (Bp4A, Bp4C). The gene Bp4C was chosen as a mastersequence for comparison. The deduced nucleotide coding sequence for thegenes Bp4A and Bp4C is shown as a sequence from which the two exons ofthe genes have been spliced together at the positions normally splicedin vivo. This gives rise to the coding sequence in the mature mRNA. ThecDNA clones are aligned with the sequence of Bp4C such that onlynucleotide changes are shown. The sequences are therefore represented asvariants of a single master sequence of gene Bp4C which is shown online 1. The ATG start codon as well as the TGA or TAA stop codons areunderlined. These three cDNA clones correspond to related members of theBrassica napus microspore specific gene family a portion of which iscontained in the clone L4.

FIG. 5 shows a partial nucleotide sequence of a cDNA clone that isclosely homologous to the gene contained in the clone L10, therestriction map of which is shown in FIG. 2b.

FIG. 6 is the nucleotide sequence of the cDNA clone that is the geneproduct of clone L19, the restriction map of which is shown in FIG. 2d.

FIGS. 7A-7E. FIGS. 7A, 7B, 7C, 7D are schematic representationsdescribing the production of vectors containing the promoter andpromoter regions from clone L4. The specific examples are discussed ingreater detail below. FIG. 7E shows a schematic representation of thepromoter constructs produced as shown, schematically in FIGS. 7A to 7E.

FIG. 8 is a schematic representation describing the production ofvectors containing the promoter regions of clone L10, the details ofwhich are discussed below.

FIG. 9 is a schematic representation describing the production ofvectors containing the promoter regions of clone L19, the details ofwhich are discussed below.

FIG. 10 is a schematic representation of a restriction map of a Brassicanapus genomic clone that contains a gene (referred to as HP101) that isconstitutively expressed at high levels in all cells includingdeveloping pollen cells. The portion of the clone that was used toprovide promoter regions for the production of anti-sense RNA is shown,this construct gives rise to an anti-sense RNA that contains a region oftranscribed RNA from this gene.

FIG. 11 is a schematic representation describing the production of agene coding for a polylysine protein.

FIG. 12 is a schematic representation describing the production of ananti-sense gene specific to the intron region of clone L19 and arestorer that lacks the intron region targetted for anti-sense RNAinhibition.

FIG. 13 is a schematic representation describing the production ofclones containing versions of a ricin A chain coding region.

FIG. 14 is a histogram showing GUS activity in plants transformed withsense and anti-sense GUS genes.

FIGS. 15A-15B illustrate the procedure used for the isolation of theT-DNA gene 2 (the IamH: indole acetamide hydrolase gene) of theAgrobacterium tumefaciens Ti plasmid derivative pPCV 311 (described byKoncz, C. and Schell, J., Molecular and General Genetics, 1986,204:383-396) and the construction of a promoterless version of thisgene.

FIG. 16 illustrates the procedure used for the isolation of the T-DNAgene 1 (the IamS: indole acetamide synthase gene) of the Agrobacteriumtumefaciens Ti plasmid derivative pPCV 311 and the construction of apromoterless version of this gene.

FIG. 17 illustrates hybrid seed production using the binarycryptocytotoxicity method based on the use of the IamH and IamS genesdescribed in FIGS. 15 and 16.

FIG. 18 illustrates the segregation patterns of the IamH and IamS genesin the F1 and F2 populations when the genes are on the same segregationunit.

FIG. 19 is an alternate representation of the procedure used to isolatethe T-DNA gene 2 (the IamH gene) of Agrobacterium tumefaciens and theproduction of a promoterless version of this gene.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned the invention relates to a recombinant DNAmolecule for use in the preparation of a male sterile plant or a plantcarrying a male sterile trait and/or reproductive material of theplants. The recombinant DNA molecule contains one or more DNA sequenceswhich may be the same or different, which encode a gene product. Thegene product includes a nucleotide sequence, in particular an RNAsequence, a protein or a polypeptide. When produced in a cell and/ortissue of a plant which is essential to pollen formation and/or functionthe gene product is directly or indirectly capable of interfering withthe function and/or development of the cell. A cell and/or tissue of aplant which is essential to pollen formation and/or function includescells and/or tissues ok that are instrumental in the development orfunction of pollen, including cells and/or tissues from which pollendevelops (e.g. premeiotic and uninucleate microspore cells), cellsand/or tissues which form part of the male structure in which pollendevelops (e.g. anther, tapetum or filament) and pollen itself. Therecombinant DNA molecule may also contain one or more promoters, whichmay be the same or different, which are capable of regulating theexpression of the DNA sequences in the recombinant DNA molecule. The DNAsequences and promoters in the recombinant DNA molecule are selectedsuch that the gene product selectively Interferes with the functionand/or development of a cell of a plant what is essential to pollenformation and/or function.

The recombinant DNA molecule of the invention may contain one or moreDNA sequences which may be the same or different. The DNA sequences maybe selected from the following DNA sequences:

(a) a DNA sequence which encodes an RNA which when produced in a cell ofa plant which is essential to pollen formation and/or functionsubstantially interferes with the expression of;

(i) a gene which is essential to pollen formation and/or function;

(ii) a gene which is essential to the continued development and/orfunction of all metabolically competent cells of a plant; or

(iii) a gene which confers on a cells of a plant resistance to achemical agent or physiological stress;

(b) a DNA sequence which encodes a protein or polypeptide which whenproduced in a cell of a plant which is essential to pollen formationand/or function is substantially cytotoxic to said cell; and

(c) a DNA sequence which encodes a protein or polypeptide which whenproduced in a cell of a plant which is essential to pollen formation orfunction renders a non-toxic substance substantially cytotoxic to saidcell; and

(d) a DNA sequence which encodes a gene product which when produced in acell of a plant which is essential to pollen formation and/or functionrenders said cell susceptible to a chemical agent or physiologicalstress.

(e) a DNA sequence which encodes a gene product which may be convertedinto a substance which is cytotoxic to cells/tissues essential forpollen formation and/or function.

The above DNA sequences (a) to (e) are further described below inrelation to the various embodiments of the invention.

According to a first embodiment of the present invention genic malesterility may be produced by Transforming plant cells that are capableof regeneration into a differentiated whole plant, with a recombinantDNA molecule containing an anti-sense gene which encodes a gene product,in particular RNA which is capable of interfering with the expression ofa sense gene. Preferably, the recombinant DNA molecule encodes RNA whichis complementary to and capable of hybridizing with the RNA encoded by asense gene.

An anti-sense gene is a DNA sequence produced when a sense gene isinverted relative to its normal presentation for transcription. Ananti-sense gene may be constructed in a number of different ways,provided that it is capable of interfering with the expression of asense gene. Preferably, the anti-sense gene is constructed by invertingthe coding region of a sense gene relative to its normal presentationfor transcription to allow for transcription of its complement; hencethe RNA's encoded by the anti-sense and sense gene are complementary. Itis understood that a portion of an anti-sense gene incorporated in therecombinant DNA molecule of the invention may be sufficient toselectively interfere with the expression of a sense gene and thus theterm "anti-sense gene" used herein encompasses a functional portion ofan anti-sense gene.

A sense gene is a gene which may only be expressed in cells and/ortissues of a plant that are essential to pollen formation and/orfunction. Preferably, the sense gene is a gene only expressed atspecific stages during pollen development whose regulation is tightlycontrolled. The sense gene may also be a gene that is essential for thecontinued development or function of all metabolically competentcells/tissues such as but not limited to genes involved in essentialcellular structures, and essential metabolism including essentialbiosynthesis. The sense gene further may be a gene which confers oncells/tissues of a plant resistance to a chemical agent or physiologicalstress.

It is understood herein that it is not essential to identify and isolatethe sense gene de novo. Rather, the sense gene may have been describedin the literature or obtained commercially.

The isolation of sense genes that are essential to pollen formationand/or function may be accomplished by a variety of procedures. Adetailed description of a procedure for isolating such sense genes isset out below.

Since the sense gene in question may initially be isolated in its nativeform, is to be understood that the term "sense gene" as used herein mayrefer to one or more parts of the gene including 5' untranslated leadersequences, coding sequences, promoter sequences, intron sequences, anduntranscribed 3' sequences, or any substantial fragments of thesesequences.

The present inventors have isolated DNA sequences from a plant of thespecies Brassica napus ssp oleifera w Westar which are expressed only inmicrospores and whose expression is essential to microspore functionand/or development and hence a preferred embodiment of the presentinvention provides a recombinant DNA molecule containing, one or moreanti-sense genes to these isolated DNA sequences. A schematicrepresentation of the restriction maps and coding regions of themicrospore specific genes identified as L4, L10, L16 and L19 are shownin FIGS. 2a, 2b, 2c, and 2d, respectively. The complete nucleotidesequence of clones L4, and relevant sequences of L10, L16 and L19 areshown in FIGS. 3a, 3b, 3c, and 3d respectively. The nucleotide sequencesof isolated cDNA clones that correspond to the genes or related genefamily members within clones L4, L10 and L19 are shown in FIGS. 4, 5,and 6, respectively. In a preferred embodiment of the invention therecombinant DNA molecule contains one or more of an anti-sense gene tothe microspore specific genes identified as L10 and L19.

It is believed that identical or homologous microspore specific DNAsequences to those isolated from Brassica napus described herein will befound and expressed exclusively in pollen of other species ofpollen-bearing plants, particularly species of plants within the genusBrassica and the family Cruciferae (also known as Brassicaceae), andmore particularly other cultivars of Brassica napus.

The occurrence of the microspore specific sequences isolated fromBrassica napus in other species of pollen-bearing plants may beroutinely ascertained by known hybridization techniques. It is believedthat the similarity of plant genes from species to species will allowfor the embodiments of the present invention to be carried out using theDNA sequence in any number of pollen bearing plant species that arecapable of being genetically transformed. The universality of plantgenes has been widely documented in the literature and homologous plantgenes have been described for plant actins (Shah, D. M., et al, J. Mol.Appl. Genet. 2:111-126, 1983), phytochrome (Hershey, H. P., et al.,Proc. Natl. Acad. Sci. USA 81:2332-2337, 1984) storage proteins (Singh,N. K., et al., Plant Mol. Biol. 11:633-639, 1988) enzymes such asglutamine, synthase (Lightfoot, D. A., et al, Plant Mol. Biol.11:191-202, 1988, and references within) and nitrate reductase (Cheng,C., et al, EMBO Jour. 7:3309-3314). These and other examples in theliterature clearly demonstrate that many plant genes are highlyconserved. It is also clear that this conservation applies not only tostructural proteins but to enzymatic proteins important to cellularphysiology. Therefore, it is believed that the DNA sequences, when foundin another plant species, will be essential to microspore developmentand will be useful in carrying out the present invention in suchspecies.

It has also been demonstrated that anti-sense RNA sequences derived fromone plant species can effectively inhibit the expression of homologousDNA sequences in a different species (Van der Krol, et al, 1988, Nature333:866-869). Therefore, it is expected that anti-sense RNA derived allor in part from Brassica microspore specific DNA sequences will befunctional in other plants.

As hereinbefore mentioned the sense gene may also be a sense gene thatis essential for the development and/or function of all metabolicallycompetent cells/tissues such as but not limited to genes involved inessential cellular structures, essential biosynthesis and essentialmetabolism. Examples of such sense genes may be found in the literatureand include the genes which encode actin, tubulin or ubiquitin; threeproteins which are essential to cellular growth and development.

Sequences for ac-in genes isolated from plants have been published (forexample; Baird W. V., and Meagher, R. B., EMBO J. 6:3223-3231, 1987, orShah, D. M., Hightower, R. C. and Meagher, R. B., Proc Natl Acad Sci USA79:1022-1026, 1982) and actin is known to play a critical role in normalcellular function especially during mitosis and meiosis where actinforms part of the cellular apparatus for cellular division.

The sequence for plant tubulin as also been described (Raha, D., Sen, K.and Biswas, B. B. Plant Mol Biol 9:565-571, 1987). Tubulin, like actin,is known to be important in the cellular life cycle particularly inregards to cell shape, transport and spindle formation during mitosisand meiosis.

The DNA sequence for plant ubiquitin has also been published (Gausing,K. and Barkardottir, R. Eur J. Biochem 158:57-62, 1986). Ubiquitin is aprotein involved in the turnover of cellular proteins and as such has acritical role in the regulation of specific cellular protein levels. Inaddition, ubiquitin is one of the most highly conserved proteins ineukaryotic cells. Interference with ubiquitin expression can causeabnormalities in the turnover of cellular proteins.

If any of the aforementioned proteins are not present in the cell,proper cellular function is interfered with and the cell fails todevelop properly.

It is believed that a gene that is found to be essential for thecontinued development or function of all metabolically competent cellsin one plant species will have a similar counterpart in other plantspecies, since it is generally understood that within the plant kingdomthere are genes that are nearly identical or very homologous involved inthe basic processes that control or are a result of cellulardevelopment. It is further believed that a gene which encodes a geneproduct which interferes with the expression of said gene (ie. ananti-sense gene) in one plant species will have the ability to do so inother plant species.

The tissue-specific and developmentally regulated expression of a wheatendosperm protein synthesized in tobacco plants genetically transformedwith this wheat gene has been reported (Flavell, R. B., et al, SecondInternational Congress for Plant Molecular Biology, Abstract #97). Inthat example, the wheat gene functioned in the tobacco plant in anidentical fashion to the way in which it functions in a wheat plant.Other literature clearly shows that the regulation of a specific gene,which can be in many cases complex, is maintained in transgenic plants.One example of this s the phytochrome mediated regulation of a wheatChlorophyll a/b-binding protein in transgenic tobacco (Nagy, F. et al,EMBO Jour. 5:1119-1124, 1986). In this example the light responsivespecific regulation of the wheat gene was maintained in the foreigngenetic environment. Not only do cereal genes function in a conservedmanner, but genes from other plant species that are more closely relatedmaintain functionality in heterologous genetic systems. Pea seedproteins are expressed properly in tobacco plants (Higgins, T. J. V., etal Plant Mol. Biol. 11:683-696, 1988), as are soybean seed proteins,(Barker, S. J., et al, Proc. Natl. Acad. Sci.USA 85:458-462, 1988) andpea rbcS genes (Nagy, F. et al., EMBO Jour. 4:3063-3068, 1985). Thescientific literature has numerous other examples of genes that havebeen used to genetically transform plants and those genes maintain theirability to function properly in this new genetic environment. Thereforethe conserved nature of these genes, not only in the DNA sequences whichcontrol the expression of these genes, but the actual protein structurecoded for by these genes, is similar among the plant species.

As hereinbefore mentioned, the sense gene may be a gene which confers oncells of a plant resistance to a chemical agent or a naturally occurringor artificially incurred physiological stress. Such a sense gene may benative to a plant cell or say be a foreign sense gene for example, agene from another plant species. If the gene is a foreign gene it may beintroduced into the genome of a plant cell prior to, after, orconcurrently with the anti-sense gene as will be described below.Examples of such sense genes may be identified in the literature andinclude genes which confer resistance to a herbicide such asacetolactate synthase which confers resistance to chlorosulfuron; geneswhich confer resistance to a physiological stress such as the geneencoding superoxide dismutase which actively reduces oxygen radicals;genes which confer resistance to uv light and wounding (phenyl andalanine ammonia lyase or chalcone synthase) genes which conferresistance to disease or pests (e.g. β1,3 glucanases, chitinase orproteinase) genes which confer resistance to antibiotics and certaintoxic drugs such as the gene encoding neomycin phosphotransferase IIconferring resistance to kanamycin.

The recombinant DNA molecule containing an anti-sense gene of thepresent invention additionally contains a promoter which regulates theexpression of the anti-sense gene. If more than one DNA sequencecontaining an anti-sense gene (or one or more of a gene encoding acytotoxic substance or a gene which confers susceptibility to a chemicalagent or physiological stress) is present in the recombinant DNAmolecule of the invention, the DNA sequences may be regulated by anidentical promoter or each may be regulated by a different promoter.

If the sense gene is a gene which is only expressed in cells/tissues ofa plant that are essential to pollen formation and/or function, then apromoter that functions in all, many, or a variety of cell typesincluding cells/tissues essential to pollen formation and/or functionmay be used in the construction of an anti-sense gene. An example ofsuch a constitutive promoter is CaMV 35S or preferably HP 101 which hasbeen isolated from Brassica napus as described below. Thus, the promoterthat is used may be constitutively active in all or many cell types butthe anti-sense gene whose expression is regulated by such a promoterwill only interfere with the expression of the sense gene in thosecells/tissues producing the sense gene in question i.e. cells/tissuesessential to pollen formation and/or function.

It is also possible to use an inducible promoter to regulate theexpression of the anti-sense gene. An inducible promoter is a promoterthat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible promoter to activate transcription is present in an inactiveform which is then directly or indirectly converted to the active formby the inducer. The inducer may be a chemical agent such as a protein,metabolite (sugar, alcohol etc.),a growth regulator, herbicide, or aphenolic compound or a physiological stress imposed directly by heat,salt, toxic elements etc. or indirectly through the action of a pathogenor disease agent such as a virus. A plant cell containing an induciblepromoter may be exposed to an inducer by externally applying the inducerto the cell such as by spraying, watering, heating,or similar methods.Examples of inducible promoters include the inducible 70 KD heat shockpromoter of D.melanogaster (Freeling, M., Bennet, D. C., Maize ADN 1,Ann. Rev. of Genetics 19:297-323) and the alcohol dehydrogenase promoterwhich is induced by ethanol (Nagao, R. T., et al., Miflin, B. J., Ed.Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3., p. 384-438,Oxford University Press, Oxford 1986). The inducible promoter may be inan induced state throughout pollen formation or at least for a periodwhich corresponds to the transcription of the sense gene. A promoterthat is inducible by a simple chemical is particularly useful since themale sterile plant can easily be maintained by self-pollination whengrown in the absence of such a chemical.

Alternatively, if the sense gene is a gene essential for the developmentand/or function of all metabolically competent cells/tissues or a genewhich confers on cells of a plant resistance to a chemical agent orphysiological stress, a promoter that is active only in cells/tissuesessential to pollen formation and/or function (for example, a pollenspecific promoter is preferably used to regulate the expression of theanti-sense gene.

The invention also relates to a pollen specific promoter or functionalportions thereof and chimeric promoters constructed using the pollenspecific promoter and/or portions thereof.

A pollen specific promoter is a DNA sequence which regulates theexpression of a DNA sequence selectively in the cells/tissues of a plantessential to pollen format-on and/or function and/or limits theexpression of such a DNA sequence to the period of pollen formation inthe plant.

Preferably, the pollen specific promoter is a DNA sequence correspondingto the promoter sequence in the microspore specific genes identified asL4, L10, L16 and L19 herein or a functional fragment thereof; or achimeric promoter sequence containing one or more of a promoter sequencefrom the microspore specific genes identified as L4, L10, L16 and L19 orportions of such promoter sequences. The preferred pollen specificpromoters may be used in conjunction with naturally occurring flankingcoding or transcribed sequences described herein or with any othercoding or transcribed sequence that is essential to pollen formationand/or function.

The pollen specific promoters referred to above were isolated from aplant of the species Brassica napus. It is believed that the it ispossible to use these promoters to limit the expression of a given DNAsequence to pollen formation and/or function and to a specific periodduring pollen formation and/or development from a plant of a differentspecies. The published scientific literature has clearly shown thatplant genes are universal and the plant tissue-specific promoterfragments retain their function in other species. For example, wheatendosperm promoter fragments function to give appropriate seed specificexpression in tobacco (Simpson, J. et al, EMBO Jour. 4:2723-2729, 1985)and the alcohol dehydrogenase promoter (Adh-1) from corn (Zea mays) canbe used in conjunction with other promoter fragments to give appropriateexpression in tobacco Ellis, J. G., et al., 1987, EMBO J. 6:11-16).Additionally the maize transposable element Ac is active in tobacco andother plant species (Taylor, et al., 1989, Plant Mol. Biol. 13:109-118)providing further evidence of the universality of plant gene structureand function. These examples demonstrate the equivalent tissue specificfunction of the same promoters in very widely divergent species(monocots to dicots). Research studies have shown equivalent promoterfunction in more closely related species within the same family orbetween families such as the Solanaceae and Brassicaceae.

It is appreciated, however, that refinements in promoter function may berequired for individual plants or species to maximize or modulate theappropriate timing or level of expression to carry out aspects of theinvention. Accordingly methods or the modification of promoters tomodify or improve function in various plants of different origin areprovided herein.

The pollen specific promoters obtained from Brassica napus describedherein may also be used to as probes to isolate pollen specificpromoters in other plant species.

It is noted that there is not a high degree of DNA sequence homologybetween the native pollen specific promoters from the L4, L10, L16 andL19 clones. Test data reveal that the timing and level of expression ofthese genes is not identical in pollen, but that all overlap in activityat some time. This illustrates that there are divergent gene sequencesin Brassica napus that still function as pollen specific promoters.

It is expected that one may use any number of different pollen specificpromoters to regulate the expression of a DNA sequence in a recombinantDNA molecule of the invention. In order to determine what pollenspecific promoter may be used to regulate the expression of ananti-sense gene (or other sequences which will be discussed below inregard to other embodiments of the invention) consideration is to begiven to a number of factors.

The pollen specific promoter used should be a promoter, or a modifiedform of the promoter, that is active at the appropriate time to producesufficient levels of transcribed RNA to carry out the invention. Thepollen specific promoters derived from pollen specific clones disclosedherein (namely those derived from the microspore genes identified as L4,L10, L16 and L19) that are active early in the development ofmicrospores such that gene expression takes place both during and afterthe meiotic and mitotic division of pollen mother cells. Thus, theactivity of these promoters is not limited by segregation.

When using a pollen specific promoter to inactivate a sense gene that isessential to pollen formation and/or function, as hereinbeforediscussed, it may be difficult to predict, a priori, which pollenspecific promoter or modified promoter construct will effectively blockthe Function of such a gene. It is preferable to use a pollen specificpromoter that displays a similar developmental pattern to the gene. Aconvenient method to determine when the sense gene targeted forinterference is expressed is to isolate RNA from developing microsporesat different stages and to analyze this RNA for the expression of thesense gene by the so-called Northern blot analysis. This procedure willallow for the determination of the developmental period in which thesense gene is expressed. In order to determine the developmental periodin which a pollen specific promoter gene is expressed, a similar seriesof analyses can be carried out using as a probe a reporter gene such asbeta-glucuronidase joined to the pollen specific promoter, or a nativesense gene from the same plant species from which a pollen specificpromoter is isolated and whose expression is regulated by the pollenspecific promoter. When the pollen specific promoter is isolated fromone plant and used in a different plant species the preferred method isthe use of a reporter gene joined to the promoter to determine the exactdevelopmental timing that the promoter has in that particular plantspecies.

It is understood that the activity of a pollen specific promoter,whether intended for the same or different species may be modified instructure to change or alter activity in a plant. Changes that arecontemplated include but are not necessarily limited to: addition ordeletion of sequences, orientation of upstream or downstream sequences,and the inclusion of introns or parts of the coding sequence of thepollen specific gene. The above modification may serve to increaseexpression or improve regulation of expression to targeted stages ofdevelopment.

It should be noted that the identification of a promoter region(including constitutive, inducible and pollen specific promoters) isusually defined by function rather than a set DNA sequence. Two hundred(200) nucleotide bases or less of a promoter sequence may be sufficientto maintain promoter function. It should also be recognized that someupstream DNA sequences can be arranged in opposite orientations andstill retain or demonstrate enhanced promoter function. In addition,"enhancer-like" DNA sequences, which are usually small conserved DNAsequences ranging in size from less than 10 nucleotides to considerablylarger numbers of nucleotides may also be Inserted into promoter regionsto enhance expression.

It may also be desirable to include some intron sequences in thepromoter constructs since the inclusion of intron sequences in thecoding region may result in enhanced expression and specificity. Thus,it may be advantageous to join the DNA sequences to be expressed to apromoter sequence that contains the first intron and exon sequences of apolypeptide which is unique to cells/tissues of a plant essential forpollen formation and/or function.

Additionally regions of one promoter may be joined to regions from adifferent promoter in order to obtain the desired promoter activity.Specific examples of chimeric promoter constructs are the chimericpromoters contained in the vectors PAL 1107 and PAL 1106.

It is desirable that the pollen specific promoter (or constitutive orinducible promoter whichever is selected to construct a recombinant DNAmolecule of the invention) function so that sufficient levels ofanti-sense RNA are provided to substantially interfere with theexpression of the sense gene. Investigations of the mechanism ofanti-sense RNA inhibition of gene expression in model systems havesuggested that equal or greater than equal levels of anti-sense RNA maybe required in order to observe a significant reduction of sense geneactivity. However, in some cases it is noted that low levels ofanti-sense RNA can have a specific reduction in sense gene activity.Therefore, in some instances if the sense gene that is targeted forinactivation by anti-sense RNA is a gene that is found to be essentialfor the continued develooment or function of all metabolically competentcells, or a gene that is expressed in all cell types at a low level, anexcess of anti-sense RNA may not be required for inhibition.Additionally less than total reduction of the gene activity may be morethan sufficient to disrupt pollen development which is known to be verysensitive to many stressful conditions. Therefore, it is suggested thatthe pollen specific promoter that is used to carry out certain aspectsof this invention be chosen based on the observation that the pollenspecific promoter functions to cause the expression of any sequencesadjacent to it to be transcribed at a time that parallels or overlapsthe period of time that the sense gene sought to be inactivated isexpressed and that the levels of anti-sense RNA expressed from theanti-sense gene be of levels sufficient to inhibit the sense geneexpression, usually to mean greater than or equal to the levels of senseRNA.

By using a pollen specific promoter to regulate the expression of ananti-sense gene, it is possible to interfere with for example, normalmicrospore development in any given plant, without having first toisolate from the genomic DNA of the plant a sense gene which isessential to microspore development. Thus, a male sterile plant may beproduced where the sense gene targeted for interference is a gene thatis essential for the development and/or function of all metabolicallycompetent cells/tissues. To produce a male sterile plant, such a gene isspecifically interfered with by using a recombinant DNA moleculecontaining a pollen specific promoter to regulate the transcription ofthe anti-sense gene.

Furthermore, a male sterile plant may be produced by growing a plantwhich has a recombinant DNA molecule comprising an anti-sense gene to agene which confers on cells of a plant resistance to a chemical agent ora naturally occurring or artificially induced physiological stress and apollen specific promoter. The transcription product of the anti-sensegene will interfere with the expression of the sense gene incells/tissues essential to pollen formation and/or function. The sensegene confers on the remainder of the plant resistance to the stress.

In order to interfere with the expression of a sense gene, it ispreferred that :he anti-sense gene and sense gene be expressed at aboutthe same time. The co-incident expression of sense and anti-sense genesmay be achieved in a variety of ways using combinations of constitutive,inducible and organ specific promoters (for example, a pollen specificpromoter as hereinbefore described). However, co-incident expression maybe readily achieved by regulating the expression of the anti-sense genewith the same promoter that controls the sense gene, thereby causingboth to be transcribed in the same time frame. The concept of regulatinggene expression using anti-sense genes is described in Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews--Trends in Genetics, Vol. 1(1) 1986.

It is preferable that the recombinant DNA molecule-containing ananti-sense gene of the invention further contain one or more selectionmarker genes which encode a selection gene product which confers oncells/tissues of a plant resistance to a chemical agent or physiologicalstress, such that plant cells transformed with the recombinant DNAmolecule or plants containing such transformed plant cells may be easilyselected using a selective agent. Any effective selective agent forwhich a resistance gene has been identified may be used to selecttransformed plant cells. The selective agent used to select plant cellstransformed with the recombinant DNA of the invention could be said tofall within two broad non-mutually exclusive categories, a chemicalagent and a physiological stress. Examples of selective agents which maybe used to select plant cells are herbicides, antibiotics, toxicsubstances and plant diseases, in particular glyphosate, chlorsulfuron,kanamycin, hygromycin, methotrexate, and bleomycin, and phosphinotricin.Preferred selection marker genes which may be used in the presentinvention to facilitate selection of transformed cells include the geneencoding the enzyme hygromycin phosphotransferase conferring resistanceto hygromycin and the enzyme neomycin phosphotransferase (NPT II) whichconfers resistance to kanamycin.

Examples of selective agents which may be used to select male sterileplants produced using a recombinant DNA molecule of the inventioninclude a chemical agent, such as a herbicide, which could be used on acommercial scale. Examples of herbicides for which a resistance gene hasbeen identified which may be useful as selective agents for plants areglyphosate (described in Comai, L., Facciotti, D., Hiatt, W. R.,Thompson, G., Rose, R. E., Stalker, D. M., 1985, Nature, Vol. 317, Pages741-744), chlorsulfuron (described in Haughn, G. W., and Somerville, C.R., 1986, Mol. Gen. Genet., Vol. 210, Pages 430-434) and phosphinotricin(Murakami T, et al, Mol. Gen. Genet. 205:42-50, 1986).

Genes encoding proteins or polypeptides that can provide a distinctivephenotypic characteristic to plant cells such as a gene that encodes aprotein providing a distinguishable color or morphology to plant cellsand genes which encode proteins which confer stress tolerance such asthe metallothiones gene may also be used as selection marker genes tofacilitate the selection of transformed plants.

For a general reference on selective agents see Weising, K., Schell, J.,and Kahl, G. Ann. Rev. of Genetics, 22:421-477 (1988).

The selection marker gene may be expressed in particular cells orconstitutively in the entire plant depending on the nature of theselection gene product. The expression of the selection marker gene(s)is regulated by one or more selection marker Promoters. The selectiongene marker promoters may be isolated using techniques known in the artand include constitutive promoters such as CaMV 35S.

To facilitate selection of plant cells transformed with a recombinantDNA molecule or male sterile plants containing a recombinant DNAmolecule it is preferred that a selection marker gene be contained inthe recombinant DNA molecule, most preferably linked to the DNA sequencecontained in the recombinant DNA molecule. Where the recombinant DNAmolecule contains more than one DNA sequence it is preferred that eachDNA sequence be linked to a different selection marker gene. It iscontemplated in some embodiments of the invention that at least tworecombinant DNA molecules be used to produce a male sterile plant and insuch embodiments it is preferable that each recombinant DNA moleculecontain a selection marker gene preferably linked to the DNA sequence.

In a hybrid seed production scheme where there are alternating rows ofmale sterile plants and male fertile plants, it may be advantageous tocarry out the final selection of male steriles in the field alongsidethe male fertile donors. Therefore it is desirable if the suitable malefertile donors are previously transformed to resistance to the selectiveagent to avoid having to selectively apply the selective agent to therows of male sterile plants.

It is to be understood that a plant sought to be rendered male sterileneed not be transformed with a selection marker gene that confersresistance to a chemical agent or a naturally occurring or artificiallyinduced physiological or chemical stress if this gene is native to theplant.

The recombinant DNA molecule containing an anti-sense gene of -hepresent invention preferably also contains a termination sequence and/orother transcription regulation signals. Examples of terminationsequences which may be used in the recombinant DNA molecules of theinvention are the nos terminator found in pRAJ-221 (ClonetechLaboratories, Palo, Alto, Calif.).

A detailed description of the isolation and identification of sensegenes which are only expressed in cells/tissues of a plant that areessential are essential to pollen formation and/or function and pollenspecific promoters is set out below:

To isolate sense genes which are only expressed in cells/tissues of aplant that are essential to pollen formation and/or function and pollenspecific promoters, a genomic library of plant DNA may be constructedfrom DNA isolated from fresh young leaves according to standardmethodology (Molecular Cloning, a Laboratory Manual Maniatis, T.,Fritsch, E. F., and Sambrooks, J., Cold Spring Harbour Laboratory, ColdSpring Harbour, New York, 1982) and screened with probes derived fromseveral tissues, one of which is made from RNA specific to cells/tissuesof a plant that are essential to pollen formation and/or function(hereinafter referred to as, pollen specific RNA). The other probesshould be made from RNA from different tissues so as to represent genesexpressed in tissues of the plant that would not be expected to includegenes that are expressed in cells/tissues of a plant that are essentialto pollen formation and/or function. Examples include but are notlimited to such tissues as leaf, roots, seeds, stigma, stem and otherorgans. Some genes are expressed in all tissues and some only in arestricted number of tissues, by comparison of many plant tissues it ispossible to isolate genes expressed exclusively in cells/tissues of aplant that are essential to pollen formation and/or function.

Pollen specific RNA may be isolated from cells/tissues of a plant thatare essential to pollen formation and/or function that are at the earlyto late uninucleate stage. Although it is possible to isolate pollenspecific RNA at other stages, isolation of the pollen specific RNA maybe technically difficult at earlier developmental stages; older cellsmay have limited nuclear gene activity; and, promoters may not besuitable for use as described in the present invention. For a detaileddescription of the isolation of pollen specific RNA see Example 1Aherein.

Pollen specific RNA may be labelled for the purpose of detection. It isconvenient to make radioactive cDNA by using the pollen specific RNA andAMV reverse transcriptase in the presence of random hexanucleotideprimers and alpha-[³² P] -dCTP. Probes are used for hybridization tonitrocellulose plaque lifts of plates containing the clones of thegenomic library. Clones that can be identified as strongly hybridizingonly to pollen specific cDNA and not cDNA from any other tissue examinedare chosen. These clones are plaque purified and grown for DNAisolation. Alternative techniques for manipulation of DNA and RNA aswell as recombinant DNA, growing and isolating clones can be found instandard laboratory manuals, such as Molecular Cloning, A LaboratoryManual (Maniatis, T., Fritsch, E. F., and Sambrook, J., Cold SpringHarbour Laboratory, New York, 1982).

For applications where the genomic DNA sequence of L4, L10, L16 or L19from Brassica napus are used to carry out certain aspects of thisinvention, the preferred method to obtain a sense gene that is essentialto pollen formation and/or function is to synthetically produce ahomologous DNA sequence according to standard methodology (see Gait, M.J., Ed., {1984} Oligonucleotide synthesis, a practical approach, pp1-22, IRL Press, Oxford, U.K.), label the sequence for the purpose ofdetection and use the labelled sequence to screen a Brassica napusgenomic library produced according to the methods described.

The identity of the promoter and coding region of a given genomic cloneis determined by restriction mapping and hybridization analysis. Thismay be accomplished by hybridization of cDNA probes made from pollenspecific RNA with restriction fragments of the DNA clones immobilized onnitrocellulose. Restriction endonuclease fragments which contain boththe coding region and regions of DNA on either side of the coding regionare isolated by sub-cloning in appropriate vectors. Once isolated, it isconvenient to use techniques such as S1 mapping and DNA sequencing toobtain exact coding regions and restriction sites within the sub-clonedDNA. This analysis is easily accomplished once the polarity with respectto gene transcription is known.

In order to determine the polarity of transcription of a sense geneindividual restriction fragments may be sub-cloned in commerciallyavailable vectors such as pGEM3, pGEM4,or pGEM3Z, pGEM4Z (available fromPromega Biotech, Madison, Wis., USA). By using these vectors one is ableto generate single stranded RNA probes which are complementary to one orthe other strands of the DNA duplex in a given sub-clone. These strandspecific probes are hybridized to mRNA, in order to establish thepolarity of transcription. Among these probes, one can isolate those.probes which hybridize with and hence are complementary to the mRNA.Using this information it is possible to clearly determine from what DNAstrand of the double-stranded genomic DNA molecule the sense mRNA hasbeen transcribed.

In order to delineate and isolate the promoter DNA sequences of a givengenomic clone the pGEM series of vectors can be used for theuni-directional deletion of sequences from the individual sub-clones inhybridization-protection experiments. Detailed descriptions of theseexperimental procedures can be found in a number of laboratory handbooksand in the manufacturers technical notes supplied with the pGEM seriesof vectors. These experiments will clearly establish the promoter andcoding regions of the pollen specific genomic clones.

The sequence of individual deletions in the pGEM vectors can bedetermined by dideoxy sequencing of plasmid mini-preps as described inthe manufacturer's technical notes. Deletion sub-clones that are deletedto very near the start of transcription or specific restrictionfragments that encompass the promoter region or the promoter region andthe start of transcription are chosen for the construction of genes thatare expressed only in developing microspores of pollen bearing plants.Usually the promoter fragment is inserted upstream of a terminator suchas the nos terminator found in pRAJ-221 (available from ClonetechLaboratories, Palo Alto, Calif.) and specific restriction fragmentswhich are to be transcribed into anti-sense RNA are inserted between thepromoter and terminator sequences. The entire construct is verified bycombination of sequencing and restriction digests. The anti-sense genethus constructed and verified may be inserted in T-DNA based vectors forplant cell transformation. T-DNA vectors that contain a selectablemarker are preferred. It is to be understood that the anti-sense genecan be constructed in a variety of ways depending on the choice ofvectors, restriction enzymes and individual genes used. For example, itmay be convenient to insert restriction fragments intended to betranscribed into anti-sense RNA into a T-DNA based vector to which apromoter and terminator structure have been previously added.Alternatively, it is possible to insert a promoter fragment upstream ofa coding region and terminator that has been previously added to a T-DNAbased vector. In addition, it may be desirable in some crops not toinsert the anti-sense gene into a T-DNA based vector but rather into avector suitable for direct DNA uptake. Promoters other than pollenspecific promoters can be used and joined with specific restrictionfragments of genes and terminators provided that these promotersfunction in cells and/or tissues essential to pollen formation and/orfunction.

In accordance with a second embodiment of the invention, a method isprovided for the production of a male sterile plant by transforming aplant with a recombinant DNA molecule comprising a pollen specificpromoter as hereinbefore described, and a DNA sequence which encodes agene product which renders a cell/tissue essential to pollen formationand/or function susceptible to a chemical agent or physiological stress.Such a recombinant DNA molecule can be introduced into a plant whereinall the cells of the plant in which the recombinant DNA molecule is notexpressed are resistant to the chemical agent and/or physiologicalstress.

In accordance with a third embodiment of the invention a method isprovided for the production of a male sterile plant by transforming aplant with a recombinant DNA molecule comprising a pollen specificpromoter as hereinbefore described and a DNA sequence which encodes aprotein or polypeptide which is cytotoxic to a cell which is essentialto pollen formation and/or function. By substance which is cytotoxic toa cell is meant a substance that when active disturbs the normalfunction of a plant cell/tissue preferably leading to cell death. Any,cytotoxic substance which is known to be encoded by one or moreidentifiable DNA sequences may be employed within the scope of thisembodiment of the invention, including but not limited to ricin, abrinand diphtheria toxin. Thus, the DNA sequence may encode any substancewhich is cytotoxic to a cell that is essential to pollen formationand/or function including the genes encoding ricin, abrin and diphtheriatoxin.

In addition to DNA sequences encoding cellular toxins such as ricin,abrin and diptheria toxin, the recombinant DNA molecule of the presentinvention may encode other cytotoxic substances including degrading ordestructive enzymes such as ribonuclease, DNAse, ribozymes, lipase, orprotease, substances that disrupt or de-stabilize cytoplasmic integritysuch as polylysine or poly-proline, substances that disrupt orde-stabilize proteins or polypeptides which are essential to certainbiosynthetic pathways in plant cells or interfere with the expression ofsuch proteins or polypeptides.

In accordance with a fourth embodiment of the invention, the recombinantDNA molecule may comprise one or more DNA sequences which encode a geneproduct which renders a non-toxic substance cytotoxic to a cell/tissuewhich is essential to pollen formation and/or function. In particular,any identifiable DNA sequences encoding gene products which can converta non-toxic to a cytotoxic substance can be employed within the scope ofthe invention. Examples of such DNA sequences include the following:

a) DNA sequences which encode indole acetamide hydrolase (IamH) whichconverts naphthalene acetamide to the plant growth regulator alphanaphthalene acetic acid (NAA) which is toxic to developing pollengrains, or converts indole acetamide to indole acetic acid (IAA) whichis a plant growth regulator. One source of the enzyme IaMH is thebacterium Agrobacterium tumefaciens (Inze, D., et al, 1984, Mol. Gen.Genet. 194:265-74).

b) DNA sequences which encode the enzyme methoxinine dehydrogenase (MDH)which converts non-toxic 2-amino-4-methoxy-butanoic acid (methoxinine)to toxic methoxyvinyl glycine. One source of MDH is the bacteriumPseudomonas aeruginosa (Margraff, R., et al., 1980, Experimentia36:486).

c) DNA sequences which encode the enzyme rhizobitoxine synthase whichconverts 2-amino-4-methoxy-butanoic acid to rhizobitoxine(2-amino-4-[2-amino-3-hydroxypropyl]-trans-3-butanoic acid) One sourceof the enzyme is the bacterium Rhizobium japonicum (Owens, L. D., etal., 1973, Weed Science 21:63-66).

d) DNA sequences which encode the tissue specific beta-glucuronidaseenzyme which for example converts a non-toxic analog of glucuronic acidconjugated to a toxic substance such as chloramphenicol (Gluc-Camp) orglyphosate (N-[phosphomethyl]glycine) to cytotoxic substances such aschloramphenicol and glyphosate, respectively. Other toxic substances maybe conjugated to glucuronic acid to provide a non-toxic analog ofglucuronic acid.

Where the non-toxic substance does not occur naturally in the plant cellinto which the recombinant DNA molecule of the fourth embodiment of theinvention is to be introduced, the non-toxic substance may be introducedinto the plant cell by spraying, watering or other similar means. Thenon-toxic substance may be produced in the cell by providing in therecombinant DNA molecule having a DNA sequence which encodes a geneproduct which renders the non-toxic substance cytotoxic to the cell, orin a second recombinant DNA molecule, a second DNA sequence whichencodes the non-toxic substance. The non-toxic substance may also beintroduced into the cell by providing in the recombinant DNA moleculehaving a DNA sequence which encodes a gene product which renders thenon-toxic substance cytotoxic to the cell, or in a second recombinantDNA molecule, a second DNA sequence which encodes a gene product whichconverts a substance which is endogenous to the cell to the non-toxicsubstance. For example, a cell may contain a recombinant DNA moleculehaving a DNA sequence which encodes IamH (which converts indoleacetamide to cytotoxic levels of indole acetic acid), and a secondrecombinant DNA molecule having a DNA sequence which encodes IamS. IamSconverts tryptophan which is generally endogenous to plant cells, toindole acetamide which in turn is converted by IamH to cytotoxic levelsof indole acetic acid.

The recombinant DNA molecule comprising one or more DNA sequences whichencode a gene product which renders a non-toxic substance cytotoxic to acell/tissue which is essential to pollen formation and/or functionadditionally contains one or more promoters to regulate the expressionof the DNA sequences. If there is more than one DNA sequence in therecombinant DNA molecule the expression of the DNA sequences may beregulated by an identical promoter or the expression of each DNAsequence may be regulated by a different promoter. Preferably theexpression of the DNA sequences is regulated by a pollen specificpromoter as hereinbefore described so that the gene product whichrenders a non-toxic substance cytotoxic selectively interferes with thefunction and/or development of cells essential to pollen formationand/or function. Where the non-toxic substance is introduced into theplant cell by providing a second recombinant DNA molecule having asecond DNA sequence encoding a second gene product which converts asubstance which is endogenous to the cell to a non-toxic substance, theexpression of the DNA sequence and the second DNA sequence may beregulated by either a pollen specific promoter, an inducible promoter ora constitutive promoter so long as there is selective interference withthe function and/or development of cells essential to pollen formationand/or function. Preferably the expression of the DNA sequence encodinga gene product which renders a non-toxic substance cytotoxic isregulated by a pollen specific promoter and the expression of the secondDNA sequence encoding a gene product which converts a substance which isendogenous to the cell to a non-toxic substance is regulated by aconstitutive promoter, an inducible promoter or a pollen specificpromoter, most preferably a pollen specific promoter. For a moredetailed discussion of the above-mentioned promoters reference may bemade to the discussion of promoters in reference to the recombinant DNAmolecules containing anti-sense genes.

In accordance with a fifth embodiment of the invention, the recombinantDNA molecule comprises one or more DNA sequences which encode a geneproduct which may be converted into a substance which is cytotoxic tocells/tissues essential for pollen formation and/or function.

The recombinant DNA molecule of the second, third, fourth and fifthembodiments of the invention also preferably further contain one or moreselection marker genes as hereinbefore described and terminationsequences and other transcription regulation signals as hereinbeforedescribed.

The following description sets forth, in general terms, the steps thatcan be employed to produce plants which are male sterile or carry themale sterile trait, using the recombinant DNA molecules of theinvention. It is to be understood that these various steps may beaccomplished by a variety of different procedures. In the followingdescription of procedures, alternative ways to accomplish these stepsare disclosed. Preferred procedures may be detailed below and in theexamples. However, it is contemplated that other variations will beapparent to those skilled in the art.

The recombinant DNA molecules of the invention may be used to produce aplant which is male sterile or carries a male sterile trait, accordingto the following preferred steps:

(1) Introducing one or more of the recombinant DNA molecules, ascontemplated herein for carrying out the invention, into the genome ofone or more plant cells, of a plant selected from those species ofpollen producing plants which are capable of being geneticallytransformed, preferably cells from which differentiated whole plants maybe conveniently recovered;

(2) Selecting a plant cell into which the recombinant DNA molecule(s) isstably incorporated; and

(3) Regenerating from the selected plant cell a plant which is malesterile or carries the male sterile trait.

A male sterile plant may be preferably produced by the above-mentionedsteps by using the following recombinant DNA molecules of the inventionin step (1):

a) A recombinant DNA molecule comprising a DNA sequence which encodes anRNA which when produced in a cell of a plant which is essential topollen formation and/or function interferes with a gene which isessential to pollen formation and/or function and a pollen specificpromoter or a constitutive promoter;

b) A recombinant DNA molecule comprising a DNA sequence which encodes anRNA which when produced in a cell of a plant which is essential topollen formation and/or function interferes with a gene which isessential to the continued development and/or function of allmetabolically competent cells of a plant and a pollen- specificpromoter; and

c) A recombinant DNA molecule comprising a DNA sequence which encodes aprotein or polypeptide which when produced in a cell of a plant which isessential to pollen formation and/or function is substantially cytotoxicto the cell or renders a non-toxic substance cytotoxic to the cells,where the non-toxic substance is introduced into the cell by convertinga substance endogenous to the cell to the non-toxic substance, and apollen specific promoter.

A plant carrying a male sterile trait may be preferably produced by theabove mentioned steps by using the following recombinant DNA moleculesof the invention in step (1):

(a) A recombinant DNA molecule comprising a DNA sequence which encodesan RNA which when produced in a cell of a plant which is essential topollen formation and/or function interferes with a gene which isessential to pollen formation and/or function and an inducible promoter;

(b) A recombinant DNA molecule comprising a DNA sequence which encodes aprotein or polypeptide which confers on a cell of a plant resistance toa chemical agent or physiological stress and a pollen specific promoter;and

(c) A recombinant DNA molecule comprising a DNA sequence which encodes aprotein or polypeptide which when produced in a cell of a plant which isessential to pollen formation and/or function renders a non-toxicsubstance substantially cytotoxic to the cell, where the nontoxicsubstance is externally applied to the cell, or where the non-toxicsubstance is introduced into the cell by converting a substanceendogenous to the cell of the non-toxic substance and an induciblepromoter controls the expression of the protein or polypeptide whichconverts the endogenous substance to the non-toxic substance.

A plant carrying a male sterile trait may be rendered male sterile byexposing the plant to a sterility actuating agent which for example isan inducer in the case of (a) above, a chemical agent or physiologicalstress in the case of (b) above, or a non-toxic substance which iscapable of being rendered cytotoxic (i.e. a cryptocytotoxic substance)to a cell which is essential to pollen formation. and/or function in thecase of (c) above.

A recombinant DNA molecule may be introduced into a plant cell by anyone of a variety of known methods preferably by first inserting therecombinant DNA molecule into a suitable vector and then using thevector to introduce the recombinant DNA molecule into a plant cell. Ashereinbefore described a recombinant DNA molecule may additionallycontain a selection marker gene which encodes a selection gene productwhich confers on a plant cell resistance to a chemical agent orphysiological stress, or confers a distinguishable phenotypiccharacteristic to the cells such that plant cells transformed with therecombinant DNA molecule may be easily selected using a selective agent.Transformed plant cells thus selected can be induced to differentiateinto plant structures which will eventually yield whole plants.

The use of Cauliflower Mosaic Virus (CaMV) (Howell, S. H., et al, 1980,Science 208:1265) and gemini viruses (Goodman, R. M., 1981, J. Gen.Virol. 54:9) as vectors has been suggested but by far the greatestreported successes have been with Agrobacteria sp. Horsch, R. B., et al,1985, Science 227:1229-1231). Methods for the use of Agrobacterium basedtransformation systems have now been described for many differentspecies. Generally strains of bacteria are used that harbour modifiedversions of the naturally occurring Ti plasmid such that DNA istransferred to the host plant without the subsequent formation oftumours. These methods involve the insertion within the borders of theTi plasmid the DNA to be inserted into the plant genome linked to aselection marker gene to facilitate selection of transformed cells.Bacteria and plant tissues are cultured together to allow transfer offoreign DNA into plant cells then transformed plants are regenerated onselection media. Any number of different organs and tissues can serve astargets for Agrobacterium mediated transformation as describedspecifically for members of the Brassicaceae. These include thin celllayers (Charest, P. J., et al, 1988, Theor. Appl. Genet. 75:438-444),hypocotyls (DeBlock, M., et al, 1989, Plant Physiol. 91:694-701), leafdiscs (Feldman, K. A., and Marks, M. D., 1986, Plant Sci. 47:63-69),stems (Fry J., et al, 1987, Plant Cell Repts. 6:321-325), cotyledons(Moloney M. M., et al, 1989, Plant Cell Repts 8:238-242) and embryoids(Neuhaus, G., et al, 1987, Thoer. Appl. Genet. 75:30-36). It isunderstood, however, that it may be desirable in some crops to choose adifferent tissue or method of transformation.

It is also understood that the successful transformation and recovery ofa plant that contains these recombinant sequences may not always resultin appropriate pollen specific expression. The transformation procedureresults in the random insertion of foreign DNA such that "positioneffects" may override and suppress the activity of any introduced DNA.It is thus advisable to generate a number of individual transformedplants with any recombinant construct in order to recover individualsfree from any limiting position effects. It may also be preferable toselect plants that contain more than one copy of the introducedrecombinant DNA molecule such that high levels of expression of therecombinant molecule are obtained.

It is known that the number of species of plants that have beensuccessfully genetically transformed still represents a modestpercentage of the total number of plant species that are of potentialcommercial interest. It is true, however that the number of species thathave been transformed has increased steadily and there is every reasonto expect that transformation systems can be developed for any crop ofinterest in due course. Routine transformation was initially achievedwith species from two plant families: Solanaceae and Brassicaceae.Examples of species of commercial interest from within these familiesthat have been transformed include: tobacco, Nicotiana tabacum L.tomato, Lycopersicon esculentum Mill, potato, Solanum tuberosum L., andpetunia, Petunia hybrida (Solanaceae); Canola/Rapeseed, Brassica napusL., cabbage, broccoli, kale etc., Brassica oleracea L., mustards,Brassica juncea L., Brassica nigra L., and Sinapis alba L.(Brassicaceae).

Recently transformation has been reported of commercially importantspecies from other families such as sugar beet, Beta vulgaris,(Chenopodiaceae), cucumber, Curcurbita sp. (Curcurbitaceae), cotton,Gossypium sp., (Malvaceae), sunflower, Helianthus annuus and lettuceLactuca sativa, (Asteraceae=Compositae), and pea, Pisum sativum,soybean, Glycine max and alfalfa, Medicago sp (Fabaceae=Leguminoseae).Transformation has also been achieved with tree species such as poplar,Populus sp. (Salicaceae) and walnut, Juglans nigra, (Juglandaceae).

Transformation success with monocotyledonous species has not progressedas rapidly, since these species are generally not very susceptible toAgrobacterium mediated transformation. However, progress which has beennoteworthy includes asparagus, Asparagus officinalis; gladiolus,Gladiolus sp., (Lilaceae); corn, Zea mays and rice, Oryza sativa(Poaceae). The recent discovery that transformation with Agrobacteriumcan be accomplished by infecting germinating seeds without therequirement of regeneration from cell culture (Chee, P. P., et al.,1989, Plant Physiol. 91:1212-1218) opens new horizons for species thatmay be difficult to regenerate. Additionally, widespread studies on theuse of particle guns to transfer microprojectiles coated in DNA intoplant cells of species that are not readily susceptible to other methodsholds great promise. It is expected that the present invention may becarried out with any one of the above species and with any other speciesthat is capable of being genetically transformed.

The most widely used and generally successful methods of introducingforeign DNA into plants are dependent upon the use of an infectiousagent, such as the Agrobacterium tumefaciens Ti plasmid, as a vector fordelivery of the foreign DNA as hereinbefore described. However, it maybe possible to use other methods, as well. Other methods that have beenemployed involve mechanical means such as direct DNA uptake, liposomes,electroporation (Guerche, P. et al, 1987, Plant Science 52:111-116) andmicro-injection (Neuhaus, G., et al, 1987, Theor. Appl. Genet.75:30-36). Recently the possibility of using microprojectiles and a gunor other devise to force small metal particles coated with DNA intocells has received considerable attention (Klein, T. M. et al., 1987,Nature 327:70-73). To date success with this and other mechanicalmethods has not been widely reported. The method of choice will bedependant on the particular plant species and will be apparent to thoseskilled in the art. In those plant species where a successfultransformation has not yet been demonstrated, it is anticipated that anynew method of transformation developed may be used to insert therecombinant DNA molecules of the invention and the actual method ofinsertion of the genes will have little or no effect on the functioningof the crop production systems described herein.

It may also be possible to produce plants which are male sterile orcarry the male sterile trait by fusing cells of a plant cell linecontaining cells having one or more recombinant DNA molecules of theinvention with cells of plant species that cannot be transformed bystandard methods. A fusion plan: cell line is obtained that carries agenetic component from both plant cells. Fused cells that carry therecombinant DNA molecule can be selected and in many cases regeneratedinto plants that are male sterile or carry the male sterile trait.

It is contemplated that some embodiments of the present invention mayrequire that a plant cell be transformed with a recombinant DNA moleculecontaining at least two DNA sequences or be transformed with more thanone recombinant DNA molecule. The DNA sequences or recombinant DNAmolecules in such embodiments may be physically linked, by being in thesame vector, or physically separate on different vectors. A cell may besimultaneously transformed with more than one vector provided that eachvector has a unique selection marker gene. Alternatively, a cell may betransformed with more than one vector sequentially allowing anintermediate regeneration step after transformation with the firstvector. Further, it may be possible to perform a sexual cross betweenindividual plants or plant lines containing different DNA sequence orrecombinant DNA molecules preferably the DNA sequences or therecombinant molecules are linked or located on the same chromosome, andthen select from the progeny of the cross, plants containing both DNAsequences or recombinant DNA molecules.

Where at least two recombinant DNA molecules are necessary to interferewith the function and/or development of a cell of a plant that isessential to pollen formation and/or function (e.g. a first recombinantDNA molecule contains a first DNA sequence encoding a first gene productwhich converts a non-toxic substance to a cytotoxic substance and thenon-toxic substance is introduced into the cell by means of a secondrecombinant DNA molecule which contains a second DNA sequence whichencodes a second gene product which converts a substance endogenous to aplant cell to the non-toxic substance), the recombinant DNA moleculesmay be linked to prevent segregation of DNA sequences required toproduce the desired effect. However it is preferred that a male sterileplant line be produced by crossing two plant lines each containing oneof the recombinant DNA molecules which are not linked, which plant linesare otherwise isogenic. Each plant line is preferably made homozygous,for the respective recombinant DNA molecules to ensure that all theprogeny receive a copy of each of the recombinant DNA molecules.Further, in some hybridization schemes discussed below, it may bepreferable, to have the respective recombinant DNA molecules located inthe same chromosome pair in each line. The choice of a chromosome paircontaining a first recombinant DNA molecule in one plant line may bepredetermined, randomly, and the other plant line may be adjusted sothat a second recombinant DNA molecule is located on the same chromosomepair. For example, to produce a plant line which is homozygous for afirst recombinant DNA molecule, a chromosome pair, in which a secondrecombinant DNA molecule is located is identified in a plant line, atransformed cell in which the first recombinant DNA molecule isincorporated on the identified chromosome pair is then selected, a plantis regenerated from the transformed cell, the plant is selfed, and aplant is selected which is homozygous for the trait encoded by the firstrecombinant DNA molecule, and the number of homozygous plants areincreased, by selfing in isolation.

Methods of identifying the chromosome pair in which a recombinant DNAmolecule is located and methods for producing homozygotes are discussedin detail below.

As indicated above, it may be desirable to produce plant lines which arehomozygous for a particular gene. In some species this is accomplishedrather easily by the use of anther culture or isolated microsporeculture. This is especially true for the oil seed crop Brassica napus(Keller and Armstrong, Z. Pflanzenzucht 80:100-108, 1978). By usingthese techniques, it is possible to produce a haploid line that carriesthe inserted gene and then to double the chromosome number eitherspontaneously or by the use of colchicine. This gives rise to a plantthat is homozygous for the inserted gene, which can be easily assayedfor if the inserted gene carries with it a suitable selection markergene for detection of plants carrying that gene. Alternatively, plantsmay be self-fertilized, leading to the production of a mixture of seedthat consists of, in the simplest case, three types, homozygous (25%),heterozygous (50%) and null (25%) for the inserted gene. Although it isrelatively easy to score null plants from those that contain the gene,it is possible in practice to score the homozygous from heterozygousplants by southern blot analysis in which careful attention is paid tothe loading of exactly equivalent amounts of DNA from the mixedpopulation, and scoring heterozygotes by the intensity of the signalfrom a probe specific for the inserted gene. It is advisable to verifythe results of the southern blot analysis by allowing each independenttransformant to self-fertilize, since additional evidence forhomozygosity can be obtained by the simple fact that if the plant washomozygous for the inserted gene, all of the subsequent plants from theselfed seed will contain the gene, while if the plant was heterozygousfor the gene, the generation grown from the selfed seed will containnull plants. Therefore, with simple selfing one can easily selecthomozygous plant lines that can also be confirmed by southern blotanalysis.

Two techniques may be used to produce plant lines which carry genes thatsegregate in a similar fashion or are on the same chromosome or a set ofchromosome pairs. One may be a simple crossing strategy in which twotransformants that are homozygous for a single inserted gene are crossedto produce F1 seed. The progeny plants from the F1 seed (F1 plantgeneration) may be crossed with a recipient plant and the segregation ofthe two inserted genes is determined (F2 plant generation). For example,where the IamH and IamS genes are the inserted genes, the F1 plantsgrown from the F1 seed will be male sterile. If the originaltransformants are homozygous for a single inserted gene, when crossedwith a non-transformed plant to produce F2 seed, the F2 plants will be100% male fertile if the two transformants originally used for theproduction of the F1 seed carried the IamH and the IamS genes on thesame chromosome or in the same linkage group. If the genes are inseparate linkage groups or on different chromosomes, a variable degreeof male sterility will be seen, in theory 25% of the plants will be malesterile if the genes segregate completely independently of each other.This approach allows for the selection of breeding lines from thehomozygous transformed plant lines that contain the IamS and IamH geneswhich will segregate substantially 100% in the hybrid seed sold forcommercial use.

An alternative strategy may make use of extensive genetic maps availablefor many commercially grown crops and the many easily scoreable markersthat are known for most linkage groups or chromosomes. In some cases,linkage groups and chromosomes may be equivalent, whereas in others,there may be more than one linkage group assigned to each chromosome.When there is a marker for each chromosome, identification of thechromosome into which the recombinant gene has been inserted isrelatively simple. A cross is made between each individual transformantand a recipient plant that allows for visualization of the marker(s).

If there are scoreable markers that have been localized to each of thechromosomes in the plant, and the markers are scoreable in thegeneration produced by this cross, one can localize the segregation ofthe inserted gene with the marker, thereby establishing the chromosomallocation of that gene. This therefore allows for the chromosomal or moreimportantly the linkage group with which the inserted gene segregates.Many crops such as corn, tomato and many cereal crops have extensivegenetic maps that allow for the identification of the chromosomecontaining the inserted gene. It is contemplated that as more detailedchromosome maps are made, especially with the use of RFLP (restrictionfragment length polymorphism) maps, the assignment of inserted genes toparticular chromosomes will easily be done for most commercial cropspecies.

As a means of confirmation, or in plant species where chromosomalmarkers are not known, it is possible to use a technique calledpulse-field electrophoresis (originally, described by Schwartz andCantor, Cell, 37:p67; 1984) to determine if different transformed plantscontain inserted genes on the same chromosome. Pulse-fieldelectrophoresis is a technique that can separate large DNA pieces, evenchromosomal size, into a reproducible pattern on a gel. When this isdone, it is possible to process this gel such that the chromosome spotscan be analyzed by southern blotting techniques, localizing the insertedgene to a chromosome spot. When the entire population of primarytransformants are analyzed in this fashion, it is a simple task tochoose the two transformants that carry the inserted genes on the samechromosome spot.

As hereinbefore mentioned, after the recombinant DNA molecule(s) isintroduced into the genome of a plant cell, a plant cell having arecombinant DNA molecule stably integrated in its genome is selected.This selection step may be facilitated by incorporating a selectionmarker gene in the recombinant DNA molecule such that plant cellstransformed with the recombinant DNA molecule may be easily selectedusing a selective agent as hereinbefore described. A preferred selectionmarker gene is neomycin phosphotransferase (NPT II) which confersresistance to kanamycin and the antibiotic G-418. Cells transformed withthis selection marker gene may be selected for by testing in vitrophosphorylation of kanamycin using techniques described in theliterature or by testing for the presence of the mRNA coding for the NPTII gene by Northern blot analysis in RNA from the tissue of thetransformed plant.

Expression of the recombinant DNA molecules of the invention intransformed plant cells may be monitored using Northern blot techniques.Single stranded RNA probes which are homologous to transcripts of thecoding sequences of a recombinant DNA molecule(s) may be used to detectfor the presence of the recombinant DNA molecule in a plant cell ortissue so that the expression of the coding sequence can be ascertained.It is preferable to use agarose gel electrophoresis to separatetranscripts according to size under denaturing conditions. In the casewhere pollen specific gene expression of the coding sequence is soughtto be accomplished it is advisable to test for the expression of thecoding sequence in the cell type whose function and/or development is tobe interfered with and in other tissues, such as leaves, roots, etc., sothat tissue specific gene expression of the recombinant DNA molecule insuch cell types can be verified.

The presence of a stably integrated recombinant DNA molecule in thegenome of the plant cell may also be ascertained by using Southern blottechniques. In this procedure, total cellular or nuclear DNA is isolatedfrom the transformed plant or plant cell and preferably digested with arestriction enzyme, thereby giving rise to discrete fragments. Thesediscrete fragments may be detected in the nuclear or total DNA of thetransformed plant or plant cells by employing standard gelelectrophoresis or hybridization techniques.

The formation of microspores in plants which contain the recombinant DNAmolecule(s) of the invention and which are male sterile, is firstmonitored by visual microscopic examination of the anther structure. Asmaturation of the flower occurs, anther formation is expected to bedelayed or completely inhibited such that no mature pollen grains areformed or released.

It will be appreciated that plants produced using the method forproducing male sterile Slants of the present invention may displayvarying degrees of male sterility. This may be a result of the nature ofthe plant cell transformed or of the chance positioning of therecombinant DNA molecule in the genome of the plant cell.

The following description of the invention sets forth, in general terms,the steps that can be employed to increase the number of male sterileplants and plants carrying the male sterile trait and to producerestorer plants, hybrid seed, hybrid seed with restored fertility andhybrid seed yielding a mixture of male fertile and male sterile plants.It is to be understood that these various steps may be accomplished by avariety of different procedures. In the following description ofprocedures, alternative ways to accomplish these steps are disclosed.Preferred procedures may be detailed below and in the examples. However,it is contemplated that other variations will be apparent to thoseskilled in the art.

As hereinbefore mentioned, the invention relates to hybrid seed having agenome comprising one or more of the recombinant DNA molecule(s) of theinvention for producing plants which are male sterile or carry the malesterile trait and to seed of plants grown from the hybrid seed. Theinvention is also directed to a mixture of fertile and sterile hybridseed and to seed of plants grown from said hybrid seed. The invention isalso directed to hybrid seed having a genome comprising one or more ofthe recombinant DNA molecules of the invention for producing a malesterile plant and a corresponding restorer gene product for restoringfertility in the seed and Us seed of plants grown from the hybrid seed.

To produce hybrid seed on a commercial scale from a plant which is malesterile or carries the male sterile trait, the number of such plantsmust be increased or maintained and crossed with a suitable male fertileparent line.

A plant which carries the male sterile trait may be more readilymaintained because such plant will not self-pollinate unless it istreated with a sterility actuating agent which renders the plantsubstantially sterile. For example, in a plant containing a recombinantDNA molecule having a DNA sequence which encodes a gene product thatrenders a cell susceptible to a chemical agent or physiological stressand having a pollen specific promoter, the plant will be renderedsubstantially male sterile by treating the plant with an actuatingagent, namely the chemical agent or physiological stress. Accordingly,plants which carry the male sterile plant are preferably maintained byselfing, selecting from the selfing progeny a plant which is homozygousfor the male sterile trait, and increasing the number of plants whichare homozygous for the male sterile trait by selfing in isolation over anumber of generations. Selection for a plant which is homozygous for themale sterile trait may also be carried out by the procedures discussedabove.

A plant which is substantially male sterile is preferably maintained bycrossing the male sterile plant with a suitable male fertile plant,obtaining seed from plants resulting form the cross, growing plants fromthe seed and selecting the plants which are male sterile from among theplants grown from the seed. This process may be repeated over a numberof generations until the desired number of male sterile plants isobtained.

A plant which carries the male sterile trait may also be maintained bythe latter method.

To facilitate selection of male sterile plants in the "maintained"plants, the recombinant DNA molecule(s) used to produce the male sterileplant should preferably comprise a selection marker gene and preferablya section marker gene is linked to a DNA sequence(s) contained in therecombinant DNA molecule as hereinbefore described.

A suitable male fertile plant for the purpose of crossing with a malesterile plant to increase the number of male sterile may be, but is notlimited to, a plant of the same inbred line from which the male sterileplant is derived. In some instances referred to below, maintenance ofthe male sterile line can be produced simply by selfing in isolation.

According to another scheme the number of male sterile plants may beincreased by clonal propagation using tissue explants thereof, or otherin vitro propagation techniques.

Where cost is warranted, and maintenance cannot be readily accomplishedas discussed above, transformed plant cells may be grown in cultureaccording to routine methodology to produce a cell line. A cell line maybe regenerated according to routine methodology to increase and maintainthe male sterile cell line. Routine methods for culturing cell lines andregenerating transformed plants from cell lines is described in standardplant tissue culture hand books. (Plant Tissue and Cell Culture, Green,C. E., Somers, D. A., Hackett W. P., and Biesboer, D. D. Eds, 1987, AlanR. Liss, Inc., New York, Experiments in Plant Tissue Culture, Dodds, J.H. and Roberts, L. W. Eds, 1985, Cambridge University Press, or CellStructure and Somatic Cell Genetics of Plants, Vasil, I. K., Scowcroft,W. R., and Frey K. J., Eds., 1984, Academic Press, New York, Handbook ofPlant Cell Culture, Vol 1-4, Evans, D. A., Sharp, W. R., Ammirato, P.V., and Yamada, Y. Eds.,1984-1986, Macmillan, New York, Bio-technologyin Agriculture and Forestry, Vol 1 and 2, Bajaj, Y. P. S. Ed., 1986,Springer-Verlag, Berlin, or Plant Propagation by TissueCulture--Handbook and Directory of Commercial Laboratories, George, E.F., and Sherrington, P. D., 1984, Eastern Press, Reading).

Production of hybrid seed may be accomplished by pollination of malesterile plants with pollen derived from selected male fertile plants.Pollination can be by any means, including but not limited to hand, windor insect pollination, or mechanical contact between the male fertileand male sterile plant. For production of hybrid seeds on a commercialscale in most plant species pollination by wind or by insects arepreferred. Selection of plants for pollen donation is determined bystandard crossing of different plants with subsequent analysis of theprogeny and selection of lines with the best combining ability andsuperior agronomic traits. Restoration of fertility in the hybrids maybe accomplished by using the methodology detailed below.

For certain crops of interest, such as vegetables, it may only be theleaves, stems or roots of the plant that are sold commercially.Therefore, even though the recombinant DNA molecule(s) which renders theplant male sterile may be inherited and expressed in the hybrid plant itis not necessary to overcome or restore male fertility in the seed ofthe hybrid plant. However, for other crops, the commodity of commercemay be the seed or fruit produced by the hybrid plant. Thus for optimalcommercial utility of the hybrid it may be desirable to produce hybridseed that is fertile.

The invention contemplates a variety of recombinant DNA molecules thatmay be used to produce a male sterile plant. In a scheme where any suchrecombinant DNA molecule(s) consists of a single gene or linked genes,the gene(s) may segregate as a unit to produce a mixture of sterilehybrid seed and fertile hybrid seed. Similarly unlinked recombinantmolecules may segregate to produce a mixture of fertile and sterile seedexcept, as discussed below, where the molecules are located on differentchromosomes of the same chromosome pair. In outcrossing species, theplants which grow from the fertile seed present may allow fullpollination of the male sterile hybrid plants. Therefore, it may bepreferable to adopt a scheme to produce hybrid seed which is fullyfertile. However in species which are relatively weakly out-crossing,male sterile hybrid plants will not be fully pollinated, therebyreducing the seed yield. Therefore, where the commodity of commerce isthe seed produced by the hybrid plant, it may be desirable to producehybrid seed which is fully fertile.

The invention contemplates methods for restoring fertility in hybridplants produced in accordance with the methods of the invention.Specifically, the invention contemplates a method for restoring hybridplants produced in accordance with the methods of the invention byincorporating into a male parent plant, a restorer recombinant DNAmolecule containing a restorer gene which encodes a restorer geneproduct which compensates for a gene function that has been compromisedby a gene product(s) encoded by a first recombinant DNA molecule(s) ofthe invention, or which negates the effect caused by a gene product(s)encoded by a first recombinant DNA molecule(s) of the invention, asdiscussed below. The male parent plant thus may act as a "restorerplant". The restorer plant may be made homozygous for the gene encodingthe restorer trait to ensure that all progeny inherit a gene. Thehomozygous restorer plant may be "maintained" by selfing in isolation toproduce a "restorer line". Selection for a plant that s homozygous forthe restorer trait may be carried out, as described above, by conductinganther or isolated microspore culture of the genetically transformedplant carrying the restorer trait, or preferably by selfing the plant inisolation prior to selection.

The expression of a restorer gene may be regulated by any promoter thatis active during the period of transcription of a first recombinant DNAmolecule which encodes a gene product which substantially interfereswith the function and/or development of a cell essential to pollenformation and/or function. It is preferred that the expression of therestorer gene be regulated by the same promoter used to regulate theexpression of the first recombinant DNA molecule or any promoter that ishighly active in tissues that are essential to pollen formation and/orfunction.

Methods for restoring fertility in hybrid plants which have integratedin the genome of their cells recombinant DNA molecules of the inventionare discussed below.

The action of a, protein or polypeptide which is substantially cytotoxicto a cell of a plant which is essential to pollen formation and/orfunction, and which is encoded by a DNA sequence contained in arecombinant DNA molecule which is integrated in the genome of cells of ahybrid plant may be negated by regulating the expression of the DNAsequence encoding the protein or polypeptide. For example, an anti-sensegene to the gene encoding the protein or polypeptide may be incorporatedinto a male parent line.

In particular where the recombinant DNA molecule has a DNA sequencewhich encodes a protein or polypeptide which is substantially cytotoxicto a cell of a plant which is essential to pollen formation and/orfunction, the male sterile plant may be crossed with a suitable malefertile plant that has been previously transformed with a restorerrecombinant DNA molecule. The restorer recombinant DNA molecule maycontain a restorer DNA sequence which is in the anti-sense orientationto that of the DNA sequence encoding the cytotoxic protein orpolypeptide and a promoter controlling the restorer DNA sequence whichactivates transcription of the restorer DNA sequence at about the timeof transcription of the DNA sequence. The restorer gene product inhibitsthe expression of the DNA sequence encoding the protein or polypeptidein the hybrid plant. The promoter controlling -he expression of therestorer DNA sequence is preferably the same pollen specific promoterthat controls the expression of the cytotoxic protein or polypeptides.Inducible and constitutive promoters may also be advantageously used tocontrol the expression of the restorer DNA sequence.

A cytotoxic method of producing male sterile plants may involve thesynthesis of proteins or polypeptides capable of substantiallyinterfering with the function and/or development of a cell/tissue whichis essential to pollen formation and/or function. Methods for restoringfertility in male sterile plants produced by cytotoxic methods involvethe syntheses, in cells/tissues that are essential to pollen formationor function, of restorer gene products that specifically block,neutralize or destroy the proteins or polypeptides. If the recombinantDNA molecule integrated in the genome of a hybrid plant has a DNAsequence which encodes a cellular toxin, a restorer plant containing DNAsequences coding for a detoxifying molecule may be used to restorefertility. If the recombinant DNA molecule integrated in the genome of ahybrid plant has a DNA sequence which encodes a destructive enzyme, arestorer plant containing DNA sequences coding for a specific enzymeinhibitor may be used to restore fertility. If the recombinant DNAmolecule integrated in the genome of a hybrid plant has a DNA sequencewhich encodes a cytoplasmic disrupting molecule, a restorer plantcontaining DNA sequences encoding a specific peptidase may be used torestore fertility. Specific examples of cytotoxic proteins orpolypeptides and their restorer gene products are the proteinase enzymetrypsin and soybean or cowpea trypsin inhibitor; ribonuclease and aribonuclease inhibitor; or a starch degrading enzyme such asalpha-amylase and an alpha-amylase inhibitor.

Fertility in a hybrid plant containing in the genome of its cells arecombinant DNA molecule having an anti-sense gene may be restored asfollows. An anti-sense gene contained in a recombinant DNA molecule tobe incorporated in the genome of a plant may be selected such that itcontains sequences of a sense gene which are transcribed, but nottranslated, in an anti-sense orientation. The transcribed butuntranslated sequence may include an untranslated 5' leader sequence,intervening sequences and an untranslated 3' sequence, or anysubstantial fragments of these sequences. It is to be understood thatthe sequences or fragments thereof may be naturally occurring or foreignsequences.

Thus, any hybrid plant produced from a cross with a plant containingsuch an anti-sense gene may be restored by crossing with a restorerplant that has been transformed with a modified form of the sense genewhich does not contain the regions complementary to the anti-sense geneand therefore is not subject to the ante-sense regulation.

As described above, a plant carrying a male sterile trait may beproduced by integrating various recombinant DNA molecules of theinvention into the genome of a plant cell and regenerating the plantfrom the plant cell. The development and/or function of cells that areessential for pollen formation and/or function in the plant areinterfered with only after the plant is exposed to a sterility actuatingagent such as an inducer, a toxic agent or a cryptocytotoxic substance.Thus, restoration is inherent in growing plants produced from hybridseed in the absence of the sterility actuating agent.

Where more than one recombinant DNA molecule of the invention is used toproduce a male sterile plant, the recombinant DNA molecules may beinserted in the same chromosome pair in separate isogenic plant lines.The respective lines are preferably made homozygous for the respectiverecombinant DNA molecule(s)/gene prior to crossing the lines to producea male sterile plant. Where a first and a second recombinant moleculeare integrated into the same chromosome in the isogenic plant lines, across of these lines results in the first and second recombinant DNAmolecules being located on separate chromosomes of the same chromosomepair in the male sterile plant. Consequently, when the male sterileplant is crossed with a suitable male fertile plant of a different line,both chromosomes of the chromosome pair segregate into separate F1progeny with the result that the first and second recombinant DNAmolecules are not expressed in the same plant. Thus, the F1 hybrid seedis fully fertile. If the two recombinant DNA molecules are integratedinto different chromosomes in the male sterile plant, then a portion ofthe F1 hybrid seed will be male sterile since there is a 25% probabilityof co-segregation of the chromosomes containing both recombinant DNAmolecules into the male sterile plant. This latter approach may beadvantageous with respect to outcrossing species. When the F1 malefertile plants outcross, a portion of the F2 seed will inherit bothchromosomes containing the first and second recombinant DNA moleculesand consequently will be male sterile. Where the seed is -he commodityof commerce, it is advantageous for seed producing companies to use ascheme for hybrid seed production, where the saving of F1 hybrid seed isdiscouraged. The outcrossing in the F1 hybrid plants results in partialmale sterility in the F2 generation, thereby reducing the seed yield ofF2 plants, which is commercially desirable. An example of this method isas follows: a first male sterile plant line incorporating in its genomea recombinant DNA molecule having an IamH gene encoding IamH whichconverts non-toxic IAM to toxic levels of IAA, may be crossed with asecond plant line having a genome incorporating a second recombinant DNAmolecule having an IamS gene which converts tryptophan to IAM.

In a preferred method of the invention for producing hybrid seed a firstmale sterile plant line having a genome incorporating a recombinant DNAmolecule having a first DNA sequence encoding a protein or polypeptidewhich renders a non-toxic substance substantially cytotoxic to a cell ofa plant which is essential to pollen formation and/or function and apollen specific promoter is crossed with a second plant line whichcontains a second recombinant DNA molecule having a second DNA sequencewhich encodes a second gene product which converts a substance which isendogenous to a plant cell to the non-toxic substance. Preferably, thefirst and second plant lines used in this method are isogenic and eachline carries a homozygous loci for the first DNA sequence or the secondDNA sequence. Most preferably the first and second DNA sequences arelocated on the same chromosome pair of the plant lines, such that in anycross of the two lines a single chromosome pair contains both the firstand second DNA sequences. The first plant line is made male sterile byexposing the first plant line to the non-toxic substance. The protein orpolypeptide encoded by the recombinant DNA molecule incorporated in thegenome of the first plant line will render the non-toxic substance toxicin cells of the plant which are essential to pollen formation and/orfunction, thus producing a male sterile plant line. The male sterile,plant line also preferably has a selection marker gene linked to thefirst DNA sequence encoding the protein or polypeptide which renders anon-toxic substance cytotoxic to facilitate harvesting of the seedshaving cells containing the first and second DNA sequences.

When the first male sterile plant line and the second plant line arecrossed, the first male sterile plant line produces seeds having cellscontaining the DNA sequence encoding the non-toxic substance (e.g. IAM)and the DNA sequence encoding the protein or polypeptide (IamH) whichrenders the non-toxic substance cytotoxic (e.g. IamH converts IAM totoxic level of IAA). The seed having cells containing the first andsecond DNA sequences will produce male sterile plants which may bepollinated with a male fertile line to produce commercial hybrid seed.If the first and second DNA sequences are located on the same chromosomeor in the same linkage group, the DNA sequences will segregate completedin the F1 hybrid seed and the hybrid seed will be substantially malefertile.

Advantage is taken in the above-mentioned preferred method of the factthat most plant species produce, per plant, many hundreds of seeds. Inoilseed Brassica for example, one plant, under normal conditions canproduce one thousand seeds. Using the method described above, one canexpect a thousand-fold increase in seeds per unit area sprayed with thenon-toxic substance. That is to say that, for example, when two isogeniclines are produced that carry the IamS and IamH genes, the firstpre-production step involves the use of NAM to cause male sterility inthe plant line that carries only the IamH gene. When cross pollinatedwith the pollen from the plant that contains the IamS gene, one canexpect up to one thousand seeds per unit area, each seed capable ofgrowing into a male sterile plant. When these seeds are planted andcrossed with a male sterile plant, one can expect one thousand seeds perunit area. Therefore, if one were to plant one acre of the plant linecarrying the IamH gene and the pollinator carrying the IamS gene, thisacre would need to be sprayed with NAM. From this one acre however,enough seed would be obtained to grow 1000 acres of male sterile plantsand pollinators, and from these 1000 acres, enough hybrid seed withrestored fertility would be obtained to plant 1,000,000 acres of hybridcrop. The amount of management required to produce this hybrid seed isreduced over conventional methods because of the pre-productionamplification step employed. If the IamH gene is linked to a herbicideresistance gene, one can plant the fields randomly to ensure high ratesof cross pollination and use the herbicide to kill the pollinator plantsafter flowering. This method therefore allows for efficient hybrid seedproduction over methods where hybrid seed is harvested directlyfollowing the first cross pollination.

A particularly preferred embodiment of the above method is described inmore detail below with reference to FIGS. 17 and 18. As illustrated inFIG. 17, the method employs two plant lines which are homozygous,respectively, for the IamH gene (plant line A2) and the IamS (plant lineA1) genes and otherwise isogenic. These genes are located on the samechromosome pair of the plant. Accordingly, plants produced from a crossof these two isogenic lines will contain the IamS and the IamH generespectively on different chromosomes of a single chromosome pair. Thiswill ensure that the two genes will segregate when this plant is crossedwith a male fertile plant. To produce the hybrid seed, a two stepprocedure is used. The first step involves a pre-production of anisogenic male sterile line, the second step in the hybrid seedproduction itself. To accomplish the first step the following approachis used: The two isogenic lines A1 and A2 are planted in rows as shown,and when flowering starts, the plots are sprayed with NAM (naphthaleneacetamide). This chemical is a non-toxic version of the plant growthregulator NAA, and the action of the IamH gene converts NAM to NAA.Under the control of the pollen specific promoter, the IamH gene only,is expressed in pollen of the A2 line, and as such NAA is only made inpollen of the A2 line. Since NAA is a plant growth regulator, normalanther and microspore development is altered, leading to male sterilityin the A2 plant line when treated with NAM. The plants which contain theIamS gene under the control of the pollen specific promoter (plant lineA1) are not affected by the NAM, since they are unable to convert NAM toNAA, therefore these plants remain fully male fertile and can crosspollinate the A2 plants which have now become male sterile aftertreatment with NAM. On the A2 line, seed is produced that contains boththe IamH and IamS genes under the control of pollen specific promoters(plant seed A2/A1). The seed produced on the A2 line (plant seed A2/A1)is harvested. This harvesting can be done by harvesting specific rows.Alternatively, the IamH gene (A2) may be linked to a gene for herbicideresistance so that the herbicide can be used for the roguing of theplant line A1. Herbicide application takes place after flowering andwill kill the A1 plants so that only seed that has the genotype A1/A2 isproduced. The seed harvested from such a field will producesubstantially 100% male sterile plants. The cross produces plants thatexpress both the IamS and the IamH genes only in the pollen. This leadsto the conversion of tryptophan, an amino acid normally found in plantcells to IAM (indole acetamide) via the IamS activity and finally to IAA(indole acetic acid) via the IamH activity. The molecule IAA is a plantgrowth regulator not normally found in substantial quantities in thedeveloping pollen grains or anther of the plant. Since the IAA and theprecursor Iam are small molecules that can be transferred from cell tocell via diffusion or active transport, altered growth regulator levelsare seen throughout the anther. This altered growth regulator levelleads to abnormality in pollen and anther development, producing a malesterile plant. This plant can be pollinated with a male fertile lineleading to commercial hybrid seed. For the production of hybrid seed,the male sterile isogenic line can be planted in rows along side of asuitable male fertile plant, and the hybrid seed produced on the malesterile plant can be harvested. If the IamH gene is linked to aherbicide resistance gene, harvesting of hybrid seed is facilitated byusing the herbicide to eliminate pollinator plants after crosspollination. The entire field can then be combined. All seed producedwill therefore be hybrid. If the two genes (IamS and IamH) are locatedon the same chromosome or in the same linkage group, these two geneswill segregate completely in the F1 hybrid seed. Since the plants willcontain either the IamS or the IamH gene, but not both, the seedproduced by this hybrid cross will be substantially 100% male fertile.Therefore the plants grown from the seed of this cross will be fullyfertile and set normal levels of seed. The F2 seed that results from theharvest of this field however will contain a variable degree of malesterility, since in theory 12.5% or 2 out of 16 of the plants grown fromthis F2 seed will contain both the IamS and the IamH genes, asillustrated in FIG. 7. Therefore, particularly in poorly outcrossingspecies, the F2 generation will not be attractive to plant because up to12.5% of the plants will not sat seed. Similarly, in outcrossing specieswhich are not fully pollinated, a corresponding reduction in yield willbe seen.

It is contemplated that as a variation of the above particularlypreferred method, a number of different ways of producing the toxicmolecule specifically in pollen can be envisioned. In all approaches, atleast one step in the production of the cytotoxic molecule has to takeplace specifically within the pollen cells or anthers. For instance, itis possible to use a constitutively expressed IamS gene in a plant andto subsequently cross that plant with a plant that contains the IamHgene under the control of a pollen specific promoter such that Iam isproduced in all cells of the plant, but the growth regulator IAA isproduced only in pollen cells due to the action of the pollen specificIamH gene. Conversely, it is possible to have IamH constitutivelyexpressed in a plant, and cross this plant with a plant that contains apollen specific promoter driving the IamS gene. In this situation, thegrowth regulator IAA is only produced in pollen cells. It should becautioned that in this case, one cannot use NAM to induce transitorymale sterility in the plant that contains the IamH gene, since thatapplication of NAM would be lethal to the plant. In this case then handpollination would be the preferred way of combining those genes. Withregards to these methods the preferred embodiment of the presentinvention places both the IamH gene and the IamS gene under the controlof pollen specific promoters and preferably using the same pollenspecific promoter or a pollen specific promoter whose expressionsubstantially overlaps that of the other to each independently drive theexpression of these two genes. Additionally, by linking the IamH gene toa selectable agent such as a herbicide, hybrid seed production isgreatly facilitated. Any number of genes could be used to carry out thisinvention providing that the simultaneous production of two or moreenzymatic or synthetic activities specifically in pollen leads to theproduction of a substance which is toxic or inhibitory to normal pollengrowth or specifically interferes with anther or pollen development.This implies that one or more of these activities could be constitutivein the plant, but that the final combination of all enzyme activities belimited to pollen. It is also envisioned that one of these activitiescould be inducible by natural or artificial means such that sterilitycould be induced in plants. Specifically one embodiment of this methoduses a plant line that carries a IamS gene under the control of aninducible promoter and a IamH gene under the control of a pollenspecific promoter. These genes are preferably linked, but could beunlinked. When grown under inductive conditions, the plant becomes malesterile and can be pollinated by a suitable male fertile plant. Thesuitable plant could also carry a IamS gene under the control of apollen specific promoter such that the progeny of this cross will bemale sterile. These plants could then be crossed with a male fertileplant, producing hybrid seed. This seed would, depending on the locationand number of the inserted genes, carry varying degrees of malesterility. It is also contemplated that a suitable male fertile plant beone that carries one or more copies of an anti-sense gene that iscapable of selectively inhibiting the expression of one or more of thesegenes such that 100% fertility is recovered in the plants grown from thehybrid seed.

DETAILED DESCRIPTION OF THE FIGURES

In FIG. 1, a schematic representation of the production of theanti-sense vector PAL 1302 is shown. A plasmid containing the GUS gene(Beta-glucuronidase, described in Jefferson, R. A., Plant MolecularBiology Reporter, 1987, 5:387-405) in the anti-sense orientation flankedby the CaMV 35S promoter and the nos ter termination signal was obtainedfrom the vetor pBI 221.1 (available from Clonetech Labotatories, PaloAlto, Calif., USA). The GUS coding sequence found between the CaMV 35Spromoter and the nos ter of the vetor pBI 221.1 was excised and digestedwith the restriction enzymes Sma I and Sst I. The Sst I site was madeblunt ended using Klenow fragment of DNA polymerase I and the bluntended vector and GUS coding sequence were religated. A plasmid (pPAL303) containing the Gus coding sequence inverted with respect to thedirection of transcription of the CaMV 35S promoter was identified.

The binary vector PAL 1302 containing the anti-sense GUS gene wasconstructed using the vector pVU 1011 (obtained from The Plant BreedingInstitute, Cambridge, UK) . pVU 1011 contains the hygromycinphosphotransferase coding sequence flanked by the CaMV 35S promoter andthe nos ter inserted into the polylinker of the Agrobacterium binaryvector Bin 19 described by Bevan, M., Nucl. Acids. Res. 1984,12:8711-8721. The vector pVU 1011 can confer both hygromycin andkanamycin resistance to transformed plant cells. The insetion of theCaMV 35S promoter anti-sense GUS nos ter fragment into pVU 1011 wasaccomplished in such a way as to inactivate the NPT II gene of thisvector and was performed a follows. A small Sph I-Pst I restrictionfragment containing the right border (RB), the NOS promoter and thebeginning of the NPT II coding sequence of pVU 1011 was first subclonedinto the Sph I and Pst I sites preceeding the CaMV 35S promoter of pPAL303 to form pPAL 306. Digestion of pPAL 306 with Sph I and Eco RIreleased a fragment consisting of the RB, the NOS promoter, thebeginning of the NPT II coding sequence and the CaMV 35Spromoter--anti-sense GUS noster construct. This fragment was thenligated into the Sph I sites of pVU 1011 by adding to the ligationpGEM-4Z (Promega Biotech, Madison, Wis., USA) cut with Eco RI and Sph Ito provide a small fragment of polylinker as a bridge between the Sph Isite of pVU 1011 and the Eco RI site of the isert from pPAL 306respectively. The orientation of the insert was verified and a binaryvector (PAL 1302) possessing a reconstructed RB fragment and a NPT IIgene inactivated by the CaMV 35S promoter--anti-sense GUS gene-nosterinsertion was identified. This vector can confer only hygromycinresistance to plants and carries the anti-sense GUS gene.

In FIGS. 2a-d the orientation of the genes contained within the fourmicrospore specific clones from Brassica napus are from 5' to 3'. Asshown, the 5' region corresponds to the promoter region and isidentified with a small arrowhead. The 3' region delineates the endpoint of transcription of the gene. Clones L4, L10 and L19 were used forthe isolation of microspore specific promoter fragments and for theisolation of microspore specific coding regions. The non-transcribedregions are identified as a single thin line, while the regions of theclones that are transcribed are demarcated by a boxed area. Within thisboxed area the portion of the transcribed DNA that represents the exonregions is demarcated by being filled in black while the intronsequences are left unfilled. The approximate regions of DNA sequencedfor clones L10, L16 and L19 are shown by underlining. Restriction sitesidentified are those that are relevant to the constructs detailed below.The right and left arms of the lambda cloning vectors are not shown.

In FIGS. 3a-d, the complete DNA sequence of the clone L4 is shown alongwith the DNA sequence of the portions of the clones L10, L16 and L19that are identified in FIGS. 2a-d. In FIG. 3a, clone L4, nucleotide 1 inthe complete sequence is at the left-most Eco R1 site while nucleotide8579 is at the first nucleotide of the right-most Eco R1 site. The startof transcription of gene 1 in clone L4 is nucleotide 235. The 5' and 3'intron splice sites are identified in boldface type. The start ATG codonis shown as well as the stop termination codon. The deduced amino acidsequence of the proteins encoded for by these genes are also shown. Theend of transcription for gene 1 is approximately nucleotide 1427. Thesecond gene in clone L4 is most likely non-functional due to a insertionand a deletion that occurs in the region of the promoter and first exon.This gene was not utilized for constructs. The third gene in clone L4has a transcriptional start at position number 6298 in the DNA sequenceand transcription ends at approximately nucleotide 7490. The ATG startcodon, intron splice sites and termination stop codon are all identifiedas above. Vectors were constructed from this clone by using promoterfragments from both genes 1 and 2, as well as promoter fragments fromgenes 1 and 2 that contained the first exon and intron sequences and ashort portion of the second exon for each of the genes. The specificpromoter fragment constructs are detailed below.

In FIG. 3b, the nucleotide sequence of the region of clone L10demarcated in FIG. 2b is shown. The start of transcription is atnucleotide 1. In this sequence the ATG start codon is at nucleotides45-47, the first exon ends at nucleotide 315 the second exon starts atnucleotide 476 and extends to nucleotide 1586. The third exon starts at1673 and extents approximately to nucleotide 1989, the precise end oftranscription was not determined. The deduced amino acid sequence isalso shown. For some promoter constructs the region of the clone 5' tothe sequenced portion were used. The specific details of the constructsare listed below.

In FIG. 3c, the nucleotide sequence of clone L16 is shown. Clone L16shows considerable homology to clone L10 specifically in the portions ofthe two clones that code for protein sequence. The intron sequencesbetween the two clones differ considerably however. Clone L16 does notcontain a 5' promoter region and as such was used only as a source ofcoding sequences for anti-sense RNA constructs. Nucleotide 1 demarcatesan Eco RI site that occurs in a coding region of the DNA that ishomologous to the first exon of clone L10. By homology, this codingregion extends to nucleotide 124, where the first intron is located.This intron, which is located at the same relative position as the firstintron of clone 10, is longer than the intron in clone L10 and extendsto nucleotide 688. Nucleotide 689 is the start of the second exon andthis exon, which shows strong homology to the second exon of clone L10,extends to nucleotide 1793. There is a second intron at this point andthis intron extends to nucleotide 1909. The third exon starts at 1910and extends to aproximately nucleotide 2210. The deduced amino acidsequence is also shown for specific regions of the clone that showconsiderable homology to clone L10. The precise nucleotide wheretranscription stops has not been determined.

In FIG. 3d the nucleotide sequence of the region of clone L19 demarcatedin FIG. 2d is shown. The start of transcription is located at position 1in the sequence. The ATG start codon is at nucleotides 136-138 and thefirst intron starts at nucleotide 1201. This intron ends at nucleotide1338 wherein the second exon starts. The end of transcription occurs atapproximately nucleotide 2074. The deduced amino acid sequence is alsoshown.

In FIG. 4, the DNA sequences of three cDNA clones that are homologous tothe genes contained in the clone L4 are shown. The DNA sequence of thesethree cDNA clones as well as the sequence of the correctly splicedtranscribed regions of genes Bp4A and Bp4C in the genomic clone L4 arealigned, only the nucleotide differences within these clones are shown,nucleotides that are conserved between the sequences are only shown onthe upper sequence. The asterisks shown in FIG. 4 mark the 5' end of thecDNA clones of cBp401, cBp405 and cBp408.

In FIG. 5, the partial nucleotide sequence of a cDNA clone that ishomologous to the coding region of clone L10 is shown. This cDNA cloneis approximately 1.3 Kb in length and has Eco R1 sites at she 5' and 3'ends of the cDNA sequence that were added via synthetic linkers in thecDNA cloning procedure.

In FIG. 6, the nucleotide sequence of the cDNA clone that corresponds tothe coding region of clone L19 is shown. Identified in this sequence isthe Eco RV site present at the 5' end of the transcribed region of L19.A portion of the poly A tail is shown. Not shown are the Eco RI sitesthat were added as linkers in the cDNA cloning procedure; these sitesare present adjacent to the 5' and 3' ends of the cDNA clone.

In FIGS. 7 (a,b,c,d,e) the construction of 6 vectors containing promoterand promoter fragments from the clone L4 is described. The first step inthe construction of these vectors was accomplished by first subcloningthe Eco R1-Sst 1(nucl.1-2132) fragment containing the first gene ofclone L4 (235 base pairs of promoter/exon/intron/second exon) in thecommercially available vector pGEM-4Z(Promega Biotech, Madison, Wis.,USA) using the Eco R1-Sst 1 sites of the polylinker of this vector. Thisplasmid was named pPAL 0402. The 2.7 Kb Eco RI fragment of clone L4 thatcontains the third gene (Bp4C) was then cloned into the Eco RI site ofpGEM 4Z, leading to a plasmid called pPAL 0411. The plasmid pPAL 0402was then digested with Eco R1 and the 2.7 Kb Eco R1 fragment from pPAL0411(nucl. 5859-8579) that contains the gene number three (Bp4C) fromclone L4 was added to it. Clones were recovered that contained thisinserted 2.7 Kb Eco R1 fragment in both orientations relative to thepromoter region of the first gene. A clone that contained this thirdgene fragment in a orientation such that the promoter from the thirdgene was opposite to the promoter in the first gene was chosen andcalled pPAL 0403. The plasmid pPAL 0403 contains the entire third genefrom clone L4 oriented in such a fashion as to have the promoter regionimmediately adjacent to the 235 basepair promoter region of the firstgene in pPAL 0403. This plasmid, pPAL 0403 was digested with Dde I,producing a fragment of approximately 1.9 Kb. The Dde I sites arelocated at nucleotides 303 and 7366. Because of the orientation of thesefragments, digestion with Dde I produces a 1.9 Kb fragment. This 1.9 Kbfragment contains a copy of the third gene (Bp4C) oriented such that thedirection of transcription of this third gene is from right to left,fused to the 235 base pair promoter fragment from the first gene ofclone L4 (Bp4A) which is transcribed from left to right, ending in a DdeI site that is located 67 basepairs down stream of the major start siteof transcription and precedes that ATG start of translation codon by 2nucleotides. This 1.9 Kb Dde I fragment was made blunt with Klenowfragment and cloned into the Xba 1 site of the polylinker region of pGEM4Z previously made blunt ended with Klenow fragment. The resultantplasmid pPAL 0408, was recovered and subsequently was digested with Sal1 and Sst 1, which releases the cloned Dde 1 fragment bordered by on theleft hand side, (nucl 7366) Sal 1 and on the right hand side (nucl 303of this construct and contains a portion of the polylinker of pGEM 4Zcontaining the following unique sites: Bam HI, Sma I, Kpn I, and Sst Irestriction enzyme sites. This Sal 1-Sst 1 fragment was cloned into theSal 1-Sst 1 sites of PAL 1001. PAL 1001 is the binary vector Bin 19(described by Bevan, M., Nucleic Acids Res., 1984, 12:8711-8721) towhich has been added the nos ter polyadenylation signal as a 260 bp Sst1-Eco R1 fragment isolated from the plasmid pRAJ 221 (available fromClonetech Laboratories, Palo Alto, Calif. USA) in the Sst 1-Eco R1 sitesof the polylinker region of Bin 19. This nos ter is identified as astippled box. The binary transformation vector that resulted from theinsertion of the Sal I-Sst I fragment of pPAL 0408 into PAL 1001 wasnamed PAL 1107. The details of the construction are shown in FIG. 7a.This vector has a copy of the third gene oriented such that thedirection of transcription of this third gene is from right to left,fused to the 235 base pair promoter fragment from the first gene ofclone L4 which is transcribed from left to right, followed by apolylinker with unique sites for the insertion of DNA which consist of:Bam HI, Sma I, Kpn I and Sst I followed by the nos ter signal. Thisvector has the feature in that additional 5' non-coding sequences wereplaced upstream to the 235 base pair core promoter on Bp4A, but theseadditional 5' sequences were in a opposite orientation. The provision ofthese sequences in this orientation does not affect the pollenspecificity of the core 235 base pair promoter.

In addition to this vector, similarly structured vectors were made whichcontained essentially the same type of gene promoter arrangement butcontained the intron of the first gene (Bp4A) of clone L4. Intronsequences in plant genes have been shown in some cases to play a role ingene expression. This intron containing vector was constructed by makinga deletion series of the clone pPAL 0402. pPAL 0402 was first digestedwith Pst I and Sma I. Exonuclease III was used to unidirectionallydigest the DNA as shown (FIG. 7b). After S1 nuclease treatment andrepair with Klenow, the plasmid was relegated and clones that have haddifferent portions of the coding regions of gene Bp4A digested out ofthem were recovered. Deletion subclones were sequenced. One was chosenfor vector constructs. This is referred to as deletion 23B. Thissubclone represented a deletion that has most of the second exon of geneBp4A removed but contains the intron splice site and first exon of geneBp4A. This subclone contains a portion of the clone L4 that extends fromnucleotide 1 to nucleotide 1166. To this subclone was added the 2.7 KbEco R1 fragment from pPAL 0411 that contains the third gene of L4 (Bp4C)in such an orientation that the direction of transcription of the thirdgene is from right to left (as in PAL 1107 , pPAL 0408), fused to the235 base pair promoter region from the first gene of clone L4 which isoriented to transcribe from left to right followed by the first exon ofgene 1, the entire intron of gene 1 and 33 nucleotides of the secondexon of gene Bp4A from clone L4. This plasmid containing deletion 23Band the 2.7 Kb Eco RI fragment containing the third gene fragment wasnamed pPAL 0406. This plasmid was digested with Hind III, which yields afragment containing a small portion of the promoter of the third gene aswell as the entire promoter of the first gene, first exon, intron and aportion of the second exon. This Hind III fragment was inserted into theHind III site of PAL 1001, resulting in the vector PAL 1106 (deletion23B derived). This vector has in the following order, A portion of thepromoter from the third gene in clone L4, the entire 235 base pairpromoter of the first gene in clone L4, followed by the first exon, theintron and a portion of the second exon of gene 1 of clone L4, followedby a polylinker containing the following unique cloning sites: Sal I,Xba I, Bam HI, Sma I, Kpn I and Sst I and the nos ter polyadenylationsignal. The construct is shown in FIG. 7b.

Additional constructs with the promoter regions of the genes containedin clone L4 were done in order to provide a number of suitable vectorsthat are useful for pollen specific expression of gene sequences. Thethree genes within clone L4 (Bp4A, Bp4B, Bp4C) show very near-exact DNAhomology and this is most apparent between the first (Bp4A) and third(Bp4C) gene. The second gene (Bp4B) is a homologous copy that hasundergone sequence changes that have appear to have lead toinactivation. The extensive similarity between the first, second andthird genes in clone L4 is also maintained in the promoter region suchthat out of the first 235 nucleotides of the first and third genepromoter regions there are only 5 nucleotides that differ between them.Downstream of the TATA box in these two promoters the only differencebetween them is the presence of one additional nucleotide at the startof transcription. For example, comparison of Promoter 1, Bp4A, partiallyrepresented as: . . . TATGTTTtAAAA___ . . . with Promoter 3,Bp4C,partially represented as: . . . TATGTTTAAAA___ . . . shows that thetranscribed region underlined and the single nucleotide difference inlower case. However, within the sequence of the first gene there is anucleotide change that introduces a Dde I site (nucl 303) in theuntranslated 5' leader sequence upstream of the ATG start codon that isnot present in the untranscribed leader sequence of the third gene inclone L4. Chimeric promoter constructs were made which utilized this DdeI site in the first gene to combine with sequences from the third genepromoter. The region of the first promoter used for these constructsconsisted of the sequences contained between the Sna BI site (nucl 21)near the TATA box to the Dde I site located immediately upstream of theATG start codon in the first gene (nucleotide 303 is the firstnucleotide in the recognition sequence for Dde I). The other region ofthis chimeric promoter (5' of the TATA box) was a fragment extendingfrom the Eco R1 site of the third promoter (nucleotide 5858) to the SnaB1 site near the TATA box (nucleotide 6273). Therefore to facilitateconstruction of these pollen specific vectors, the followingreconstructions were performed.

The Eco R1 to Dde 1 fragment that encompasses the promoter region of thefirst gene in clone L4 was isolated by first cutting pPAL 0402 with Dde1, blunting with Klenow, and then cutting with Eco R1. The 235 base pairfragment corresponding to this region was cloned into the Eco R1-Sma 1sites of pGEM 4Z. This plasmid (pPAL 0422), was then cut with Eco R1 andSna B1. A DNA fragment that contained the Eco RI to Sna BI portion ofthe promoter for gene 3 in clone L4 was isolated by digesting pPAL 0411with Eco R1 and Sna B1. This released an approximately 415 base pair EcoRI (nucl.5858) to Sna BI (nucl. 6273) fragment that represents most ofthe 5' region of the gene 3 promoter from clone L4 (the Sna B1recognition site is 2 base pairs downstream of the TATA box). This EcoR1-Sna B1 fragment was used to replace the shorter Eco R1-Sna B1fragment removed for the first promoter subclone (pPAL 0422),reconstructing a promoter fragment of approximately 550 base pairs. Thisplasmid is referred to as pPAL 0421. This chimeric promoter fragmentcontains 415 base pairs of the promoter of gene three in clone L 4,followed by approximately 99 Nucleotides of the first genepromoter/untranslated leader sequence.

For construction of a pollen specific cassette vector, the followingplasmids were first constructed. The first plasmid constructed containedthe nos ter polyadenylation signal with a polylinker in front of the noster signal. This was accomplished by first isolating from pRAJ 221 thenos ter as a Sst 1-Eco R1 fragment and this fragment was cloned in pGEM4Z using the Sst 1 and Eco R1 sites in the polylinker. This subcloned isreferred to as pPAL 001. To pPAL 001, a fragment coding for neomycinphosphotransferase (NPT II) derived from the plasmid pRAJ 162 was addedto it in the anti-sense orientation as follows: The plasmid pRAJ 162contains the NPT II gene from the transposon TN 5 inserted as a Sal Ifragment and bounded by a polylinker in the plasmid pUC-9 (which wasobtained from the Plant Breeding Institute, Cambridge, UK). pRAJ 162 wasdigested with Hind III and Sma I. The DNA fragment containing the NPT IIgene was isolated by elution from an agarose gel. pPAL 001 was digestedwith Hind III and Sma I and the NPT II gene fragment was inserted. Theresultant plasmid was called pPAL 002 and had such orientation ofrestriction sites and the NPT II gene and nos ter as follows: HIND III,Pst I, Sal I, 3' end NPT II coding sequence 5' end, Sal I, Bam HI, SmaI, Kpn I, Sst I, nos ter, Eco RI. pPAL 002 was cut with Hind III and thesize made blunt ended by the use of Klenow fragment. pPAL 0421 wasdigested with Hinc II and Pvu II, both of which leave blunt ends, andthe promoter fragment was ligated into Hind III cut blunt ended pPAL002. Plasmids were obtained that contained the promoter in bothorientations relative to the nos ter signal. One plasmid was chosen withthe proper orientation (5' promoter/anti-sense NPT II/nos ter) and wasnamed pPAL 0419. pPAL 0419 has she following DNA fragments: A small(approx. 130 bp) of pGEM 4Z that contains the SP6 promoter, the 550 basepair chimeric promoter, the NPT II gene in the anti-sense orientationrelative to the promoter, followed by the nos ter polyadenylationsignal. This entire promoter/NPT II/nos ter construct is excisable byEco RI. pPAL 0419 was digested with Eco RI, and the promoter NPT II noster structure was cloned into BIN 19 using the single Eco RI site in thepolylinker of BIN 19. The resultant transformation vector was named PAL1419. In addition to the anti-sense NPT II gene, the vector contains aconstitutive NPT II gene under the control of the nos promoter. Thisvector therefore confers resistance to kanamycin in all cell types withthe exception of pollen, cells where the gene expression from theconstitutive promoter is inhibited by the anti-sense RNA produced fromthe promoter/NPT II/nos ter construct contained in PAL 1419.

In order to provide promoter sequences that could be utilized withadditional gene constructs, the plasmid pPAL 0419 was digested with SalI. This digest removes the NPT II coding region and this Sal I digestedpPAL 0149 was relegated giving rise to pPAL 0420. pPAL 0420 representsthe pollen specific promoter followed by a polylinker for insertion ofgenes that has the following unique sites: Hinc II, Pst I, Sal I, BamHI, Sma I, Kpn I, Sst I, followed by the nos ter polyadenylation signal.The entire promoter/polylinker/nos ter construct can be convenientlyexcised as a single Eco RI fragment. The details of this construct isshown in FIG. 7c.

For additional pollen specific promoter constructs, the followingapproach was used. The intact L4 clone in the lambda cloning vector wasdigested to completion with the restriction enzymes Sst I and Hha I. Theresultant fragments were separated by gel electrophoresis and a 2.65 Kbfragment that contains the promoter/first exon/intron/partial secondexon region of gene three in clone L4 and corresponds to nucleotides4565 to 7216 in the sequence of clone L4 was isolated. This fragment wasmade blunt ended with Klenow and cloned into the binary transformationvector PAL 1001 previously described. PAL 1001 was first cut with HindIII and made blunt ended with Klenow. Clones containing this fragment(promoter/first exon/intron/partial second exon) were recovered. A clonewas chosen that contained this fragment in the proper orientation suchthat the direction of transcription was towards the nos ter in PAL 1001.This vector was named PAL 1421. This vector contains approximately 1.9kb of upstream promoter region from the gene 3 in clone L4 followed bythe first exon, the complete intron and 15 bases of the second exon ofgene three followed by a polylinker containing the following uniquesites: Sal I, Xba I, Bam HI, Sma I, Kpn I, SstI, and finally the nos terpolyadenylation signal. A variant of this vector was constructed bydigesting PAL 1421 with Eco RI and isolating the fragment from thisclone that contains the promoter polylinker nos ter sequences butcontained less of the upstream region of the promoter. This fragment wasre-cloned into PAL 1009. PAL 1009 is a BIN 19 derived vector from whichmost of the polylinker has been removed. This vector was constructed bydigesting BIN 19 with Hind III and Sst I, making these sites blunt endedwith Klenow and relegating such that a vector was recovered thatcontained a single unique Eco RI site for the insertion of fragments.PAL 1009 was digested with Eco RI and the Eco RI fragment from PAL 1421that contains a shorter promoter/exon/intron/second exon/polylinker/noster structure was added to it. This gave rise to the vector PAL 1422, avector that is essentially the same as PAL 1421 with the exception thatthere is less 5' promoter region. It should be noted that both PAL 1421and PAL 1422 contain the intron from the third gene. For constructswhich the presence of the intron may not be desired, intron sequenceswere removed from PAL 1421 by first digesting PAL 1421 with Eco RI andreplacing the promoter/exon/intron/second exon/polylinker/nosterstructure with the promoter/polylinker/nos ter structure from pPAL 0420using Eco R1 such that a longer 5' promoter region is reconstructed inthe binary transformation vector. The resultant vector was named PAL1423. The outline of this construction is shown in FIG. 7d.

In FIG. 7e, a schematic diagram of the relationship of the abovedescribed vectors is presented. It should be noted that the vectorsoutlined in this Figure fall into three categories: 1, vectors whichcontain 5' upstream promoter regions that are substantially derived fromthe upstream region of the gene Bp4C (pPAL 0420, PAL 1420, PAL 1423), 2,promoter constructs that contain 5' upstream promoter regions and intronsequences from the gene Bp4C (PAL 1422, PAL 1421) and, 3, promoterswhich contain a chimeric 5' upstream region in which a portion of the 5'DNA sequence is inverted relative to the arrangement which appears inthe genomic clone and uses the promoter fragment of Bp4A as a corepromoter structure (PAL 1107 , PAL 1106). It should be noted that thefunctioning of each of these constructs can vary from plant species toplant species and it may be desirable to test a number of these promoterconstructs when carrying out certain aspects of this invention.

The construction of pollen specific vectors that utilize the promoterregions of clones L10 and L19 was conducted as follows. The constructionof the pollen specific vectors depicted in FIG. 8 utilizes promoterregions from clone L10. The start of transcription of clone L10 islocated at nucleotide 1. The ATG start codon is located at nucleotides45-47. The promoter region of this clone was excised by first subcloningthe Eco RI-Xba I fragment of the clone that encompasses the entirepromoter region and a portion of the first exon (the Xba I site isnucleotide 358 in the DNA sequence). This subclone (pPAL 10EX) was thendigested with Hinc II and Nde I. The Nde I site is located immediatelyupstream of the ATG start codon at nucleotide 60 and the Hinc II site islocated at nucleotide number -399. The digestion with these two enzymesreleases a DNA fragment of 459 nucleotides which contains 62 nucleotidesof untranslated transcribed leader sequence, and 397 nucleotides of 5'promoter region. The Nde I site in this fragment was made blunt ended bythe use of Klenow, and this fragment was subcloned into the Hinc II siteof the polylinker of pGEM 4Z. Clones were recovered in both orientationsand the clone that contained the fragment in the orientation: Hind III,Sph I, Pst I. Hinc II, promoter-62 base pair leader fragment (Nde Iblunt/Hinc II, does not cut with either Hinc II or Nde I) Xba I, Bam HI,Sma I, Kpn I, Sst I, Eco RI was chosen and named pPAL 1020. To addadditional upstream regions, the Hinc II-HincII fragment that isapproximately 1 Kb in length and is immediately upstream of the Hinc IIsite at position 391 in the DNA sequence was isolated from pPAL 10EX bydigestion with Hinc II and gel elution of this fragment. This Hinc IIfragment was cloned into the Sma I site of pGEM 4Z. Clones whichcontained the fragment in both orientations were recovered and a clonethat contained the fragment in the following orientation was chosen:Hind III, Sph I, Pst I, Hinc II, Sal I, Xba I, Bam HI, the Hinc IIfragment in the same orientation as in the genomic clone, that beingright to left, 5'-3' (as a Hinc II/Sma I insertion which does not cutwith either enzyme), Kpn I, Sst I, Eco RI. This subclone (pPAL10Hc) wasdigested with Knp I, made blunt end by the use of Klenow, then digestedwith Eco RI. To this cut subclone was added the promoter/untranslatedleader sequence of pPAL 1020 by digesting pPAL 1020 with Hinc II and EcoRI, and adding this promoter fragment to the cut pPAL 10Hc. Theresultant subclone contained a reconstructed promoter region of cloneL10 differing from the intact region by only the filled in Kpn I siteused for the joining of the two promoter fragments. This construct wasnamed pPAL 1021. This vector contains in the following order: Hind III,Pst I, Sph I, Hinc II, Sal I, Xba I, Bam HI, the approximately 1 Kb HincII fragment joined to the Hinc II-Nde I promoter fragment followed byXba I, Bam HI, Sma I, Kpn I, Sst I, and Eco RI. This subclone allows forthe convenient removal of the promoter region of clone L10 such that thepromoter can be easily used in cassette transformation vectors. Theoutline of this construction is shown in FIG. 8. The promoter region ofpPAL 1021 was used for the construction of a pollen specific cassettetransformation vector by carrying out the following constructs: Theplasmid pPAL 1021 was digested with Nco I and Pst I. The plasmid wastreated with Klenow and religated. This procedure effectively removedthe portion of the polylinker that was 5' to the promoter in pPAL 1021.This plasmid was then digested with Hind III and Sst I, and cloned intothe Hind III and Sst I sites of PAL 1001, giving rise to PAL 1121 . PAL1121 has in the following order: the pollen specific promoter of cloneL10 (approximately 1.1-1.2 Kb), followed by a polylinker with thefollowing unique sites: Xba I, Bam HI, Sma I, Kpn I, Sst I, follower bythe nos ter. The construction of this is outlined in FIG. 8.

The promoter region of the clone L19 was also used for construction ofpollen specific vectors. The construction of these vectors is as shownin FIG. 9. Clone L19 has a single pollen specific gene contained withit. The start of transcription in this gene is located at position 1 inthe DNA sequence. The ATG start codon is located at nucleotide position136-138. The only intron is located at nucleotides 1202-1387, the stoptranslation codon is located at nucleotides 2024-2026. The end oftranscription is located at approximately nucleotide 2074. The entireEco RI fragment of this clone was subcloned into pGEM 4Z by using theEco RI site located in the polylinker. The resultant clone was namedpPAL 1901. The promoter region of this clone was excised as a singlefragment by digesting pPAL 1901 with Bam HI and Eco RV, and a 2177basepair fragment corresponding to the promoter region was isolated.This fragment covers from nucleotide -2200 (Bam HI) to nucleotide 156(Eco RV). This promoter fragment contains over 2 Kb of 5' upstreamregion of the promoter in clone L19, 134 basepairs of 5' untranslatedleader sequence and 20 basepairs of translated sequence. The Bam HI sitein this fragment was made blunt ended by the use of Klenow and clonedinto PAL 1001. This step was accomplished by cutting PAL 1001 with HindIII, making this site blunt ended by the use of Klenow and inserting theblunt ended Bam HI-Eco RV fragment in such an orientation that thepromoter was oriented 5' to 3' with respect to the polylinker/nos terpolyadenylation signal. This vector was named PAL 1920 and containedwithin it in the following order: The promoter from clone L19 containing134 base pairs of 5' untranslated leader sequence, 20 base pairs oftranslated sequence fused to a polyliker containing a former Hind IIIsite inactivated by blunt ending, Sph I, Pst I, Sal I, Hinc II, Xba I,Bam HI, Sma I, Kpn I, Sst I (the unique cloning sites are underlined),the nos ter polyadenylation signal. This vector is convenient for theinsertion of DNA sequences to be transcribed in pollen cells. Theoutline of this construct is shown in FIG. 9.

In FIG. 10, the restriction map of a Brassica napus genomic clone (HP101) that contains a constitutively expressed gene is shown and thefragment of this clone that contains a 5' promoter region along with aportion of transcribed sequence is identified. This fragment wasisolated by first cloning the small 2.5 kb Eco RI fragment in pGEM 4Z,and obtaining a subclone that had this fragment inserted in theindicated orientation relative to the polylinker of pGEM 4Z. This clonewas called pPAL 0101 and was deposited Jan. 26, 1990 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.,20852, USA as pPAL0101/E. coli strain DH5 alpha under acession numberATCC 68210. This E. coli strain grows on standard E. coli media (LB)with 100 micrograms per ml of ampicillin. This subclone, pPAL 0101, wasthen digested with Eco RI, treated with Klenow fragment, then digestedwith Bam HI, which releases the promoter/transcribed region indicated.This fragment was cloned into Hinc II-Bam HI cut pGEM 4Z, resulting inthe subclone pPAL HP101. The subclone can be used for the isolation ofpromoter sequences in vector constructs that utilize a constitutivepromoter to synthesize pollen specific anti-sense RNA.

In FIG. 11 a schematic representation of producing a polylysine codinggene is shown. In this construct, the cloning vector pGEM 4Z was used asa recipient of a synthetic oligonucleotide containing a ATG start codonand to this was added a polynucleotide consisting of solely onenucleotide. This gene therefore, depending on the nucleotide used is agene that has predominantly one codon, and codes for a protein that iscomposed of a polyamino acid. The construct was carried out in thefollowing fashion: To provide a ATG start codon in a favourableinitiation context, a synthetic oligonucleotide was constructed andinserted into pGEM 4Z between the Hind III and Sph I sites. Thisnucleotide had the sequence: 5'-ACGTGGATCCAAGATGACATG-3'. The resultantsubclone therefore had the DNA sequence (restriction site for aintroduced Bam HI site is underlined, the ATG start codon in bold) AACGTGGATCC AAG ATG ACA TGC GCA ACA TGG at the 5' region such that there wasa ATG start codon in a favourable initiation context and a Bam HI siteupstream of this site for excision of the coding sequence. This subclonewas digested with Pst I, divided into two aliquots, one was tailed withT residues using terminal transferase and TTP, one was tailed with Aresidues using terminal transferase and dATP. The two tailed plasmidswere mixed together, extracted with phenol-chloroform, ethanolprecipitated and resuspended. The plasmid mixture was cut with Sst I,and relegated. Clones that were recovered were either clones thatcontained on the coding strand polyA or poly T. Clones were cut with BamHI, the size of the insert determined by gel electrophoresis, andsequenced to determine if the clone coded for poly-lysine (polyA) orpoly-phenylalanine (poly T, poly U in the mRNA). A clone that coded forpoly-lysine and was approximately 300 nucleotides was chosen and calledpPAL pLys. This clone was cut with Xba I, blunt ended with Klenow and auniversal translation terminator (available from Pharmacia PLBiochemicals, Montreal, Canada) was added to it, completing theconstruction of the gene. This construction is shown in FIG. 10.

In FIG. 12 a scheme for the production of an anti-sense gene specific tothe intron region of clone L19 and the production of an intronlessversion of the number L19 gene is shown. For the first step in this, arestriction fragment from clone L19 was isolated that is substantiallyall intron. This fragment was isolated by using the restriction enzymeDde I which cuts at a number of sites in the genomic clone, but thesites at nucleotides 1186 and 1348 give rise to a restriction fragmentthat is substantially intron sequences, having only approximately 16nucleotides at the 5' side of the intron that are included in the finaltranscript, and 10 nucleotides at the 3' side of the intron that areincluded in the final transcript. This Dde I fragment was isolated bygel electrophoresis, made blunt ended and cloned into Sma I cut pGEM 4Z.Clones in both orientations were obtained, and the clone that containedthe intron region in the orientation: Hind III, Sph I, Pst I, Sal I, XbaI, Bam HI (former Sma I) 3' end of the intron, intron sequences, 5' endof the intron, (former Sma I), Kpn I, Sst I, Eco RI was chosen. Thisclone was named pPAL 1914 and was digested with Bam HI and Sst I, andinserted into PAL 1920 previously cut with Bam HI and Sst I, creatingthe vector PAL 1954.

To create a specific restorer vector, the cDNA clone 19 cDNA (FIG. 6)was fused to the promoter region as follows: The cDNA clone was digestedwith Eco RV and Sma I as shown in FIG. 11. To this cut vector was addedthe Eco RV-Sma I fragment from clone L19. Clones were recovered thatcontained the reconstructed 5' region of the promoter and codingsequence, but carried coding sequences that lacked the intron, most ofthe coding regions being derived from the cDNA clone. This clone wasdigested with Eco RI, made blunt ended with Klenow, then digested withSma I. A DNA fragment that represents the entire coding region and aportion of the promoter region was isolated and cloned into PAL 1920 cutwith Sma I, leading to a reconstruction of the promoter region andcoding region lacking an intron. This vector was called PAL 1955.

In FIG. 13, the production of clones containing the coding sequences ofa protein functionally related to the ricin A chain protein isolatedfrom Ricinus communis is detailed. This was accomplished by firstisolating a genomic clone homologous to ricin from a genomic library ofRicinus zanzabarenis DNA constructed in the vector lambda gt10 usingstandard protocols. The library was screened with a DNA probe thatcorresponds to the N-terminal leader sequence of the ricin gene. Theprobe sequence was obtained from the published sequence of a Ricinuscommunis ricin gene (Halling, et al., Nucl. Acids Res 13:8019-8033,1985). A genomic clone was isolated that contained the leader sequenceand a portion of the A chain was isolated and called RIC 1B. This clonecontained the promoter region, the 5' untranslated region, theN-terminal leader sequence and coding region that extended to amino acid191(lle) in the published sequence (Halling, et al., Nucl. Acids Res13:8019-8033, 1985). The difference between the Published sequence andRIC 1B was that the published nucleotide sequence at the region of lle191 was: (lle 191 underlined) ACG AGA ATT CGG which codes for the aminoacids: The Arg lle Ara while in RIC 1B the nucleotide sequence is ACGAGA ATT CGG, which codes for the same amino acids (The Arg lle Arg), theonly difference being the last Arg is coded for by CGG in RIC 1B whilein the published sequence it is coded for by AGG. This single nucleotidesubstitution has the effect of introducing a Eco RI site at lle 191. Theclone RIC 1B therefore was missing the amino acids present after lle 191since the clone was isolated as a single Eco RI fragment. This truncatedversion of ricin was used for construction of ricin A chain N-terminaldeletions as follows: The clone RIC 1B was digested with Hinc II and EcoRI. The fragment was cloned into Eco RI-Sma I cut pGEM 4Z. The resultingclone, pPAL R1B was digested with Pst I and Bam HI. This cut clone wasdigested with Exo III nuclease, treated with S1 nuclease and Klenowfragment and then relegated. Subclones were obtained that had variousportions of the 5' region deleted, and some of these deletions weresequenced. One deletion, named pPAL-Ridefin had the majority of theN-terminal leader DNA sequence removed and had Hind III and Sph I sites5' to this region such that the DNA sequence was as follows: AAGCTTGCATGC GCA ACA TGG . . . wherein the first six nucleotides code for aHind III site found in pGEM 4Z, the next six nucleotides codes for theSph I site in pGEM 4Z and the following three triplets code for aminoacids -20, -19, -18 . . . (Ala The Trp . . . ) in the published sequence(Halling, et al., Nucl. Acids Res 13:8019-8033, 1985). This subclonetherefore had a deletion that removed the first 15 amino acids of the Achain leader sequences of ricin. To provide a ATG start codon in afavourable initiation context, a synthetic oligonucleotide wasconstructed and inserted into the subclone between the Hind III and SphI sites. This nucleotide had the sequence: 5'-ACGTGGATCCAAGATGACATG-3'.The reconstructed clone (pPAL Rictr) therefore had the DNA sequence(restriction site for a introduced Bam HI site is underlined, the ATGstart codon in bold) AACGT GGATCC AAG ATG ACA TGC GCA ACA TGG at the 5'region such that there was a ATG start codon in a favourable initiationcontext and a Bam HI site for excision of the coding sequence. The clonepPAL Rictr was digested with Eco RI, end filled with Klenow, anddephosphorylated with alkaline phosphatase. To the vector was added theuniversal translational terminator purchased from Pharmacia-PLbiochemicals (Montreal, Canada) to provide a termination codon. Thecoding region from this clone was isolated by digestion with Bam HI andPvu II, releasing the coding region and a small portion of pGEM 4Z, andthis fragment can be cloned into the Bam HI and Sma I sites oftransformation vectors. This DNA fragment codes for a version of a ricinA chain in which a C-terminal portion has been deleted. It should benoted that a number of C-terminal and N-terminal deletions of the ricinA chain have been tested in vitro for toxicity, and these reports haveconcluded that the N-terminal half of the ricin A chain is sufficientfor cytotoxicity in vitro (for example see: Sudan et al., Nuc . AcidsRes, 17:1717-1732, 1989). In order to obtain a complete coding regionfor the ricin A chain, synthetic version of the rest of the A chain wassynthesized using the published sequence. This synthetic portion of thegene extended the DNA sequence to nucleotide 1182 in the publishedsequence and had a Eco RI sites at both ends that allowed for thejoining of this fragment to the Eco RI site at the amino acids numbers190-192. This reconstructed version of the gene also had a Bam HI siteafter the stop codon such that the DNA sequence of the gene at the 3'end was as follows: (nucl. 1181 is marked *) CCT CCA* TAA GGATCC GAATTCcoding for amino acids: Pro Pro stop. followed by Bam H: and Eco RI, theEco RI site being used for the insertion of the synthetic portion of thegene, the Bam HI being used for excision of the complete ricin A chainsequence since a synthetic Bam HI site is at the 5' end of the codingregion also. The clone was called pPAL Riccom.

In FIG. 14 the results of anti-sense RNA inhibition ofBeta-glucuronidase gene activity is shown in a histogram of GUS geneactivity found in transgenic plants that contained a sense GUS gene andwere retransformed with a vector containing an anti-sense GUS gene. GUSactivity levels were expressed as a percentage of the GUS activity foundin the original GUS+ plant TTR-48. Tissue was obtained from young (Y),medium (M) and old (O) leaves and GUS activity was assayedspectrophotometrically. Lanes 1-5, samples from the retransformed plantsTTR-1 to TTR-5 (TTR-48/PAL 1302); lane 6, samples from plant TTR-48;lane 7, samples from TTR-88(TTR-48/pVU 1011) and lane 8, samples fromuntransformed tobacco.

EXAMPLE 1

In this example, anti-sense RNA was used specifically for the inhibitionof gene activity in plants. A tobacco plant expressing theBeta-glucuronidase gene under the control of the CaMV 35S promoter wasproduced by transformation of a non-transformed control tobaccocultivar, N. tabaccum, cv. Delgold. To accomplish this, tobacco leavesless than 8 inches in length were surface sterilized by exposure toethanol for 5-6 seconds, then subsequent exposure to 1% sodiumhypochlorite for a few minutes, usually 5-10 minutes, or until the cutedge of she petiole turned white, then rinsed several times in steriledistilled water. Leaf segments of approximately 0.5 to 1.0 squarecentimeters were cocultured for two days with Agrobacterium tumefaciensGV 3101 carrying the Ti plasmid pMP 90 to provide vir functions in trans(described by Koncz, C. and Schell J., 1986, Mol. Gen. Genet.204:383-396) carrying the binary vector pBI 121.1 on shoot inducingmedia. This vector is a derivative of Bin 19 which contains the GUS genedriven by the CaMV 35S promoter and terminated by the nos ter and isavailable from Clonetech Laboratories, Palo Alto, Calif., USA.Transformed tobacco cells were selected on a shoot-inducing mediumcontaining 0.8% agar, MS salts, B5 vitamins, 3% sucrose, 1 mg per L ofbenzyladenine, 0.1 mg per L of alpha naphthalene acetic acid, (NAA) 300ug/ml kanamycin and 500 ug/ml carbenicillin (essentially as described byHorsch et al, 1985, Science, 227:1229-1231). Regenerated shoots werethen transferred to a root-inducing medium consisting of B5 medium with2% sucrose, 500 ug/ml carbenicillin and 0.5 mg/L each of NAA andindoleacetic acid IAA). Following selection on kanamycin, a tobaccotransformant which displayed relatively high constitutive levels of GUSactivity and contained a single unrearranged insertion of the 35S CaMVpromoter-GUS-noster construct was selected. This plant (TTR-48, GUS+)was then re-transformed with a binary vector PAL 1302 which contains ananti-sense GUS gene, the construction of which is described in FIG. 1.In experiments involving the retransformation of the TTR-48 tobaccoplant with PAL 1302, the shoot-inducing medium contained 20 ug/mlhygromycin and 300 ug/ml kanamycin to insure the selection of plantscontaining both the sense and anti-sense GUS constructs. Transformantswere grown to maturity and self-pollinated in the greenhouse.

The leaves of tobacco plants resulting from the retransformation ofTTR-48 (GUS+) with the anti-sense GUS vector PAL 1302 were assayed forGUS activity. The GUS activity in leaf extracts was assayedspectrophotometrically. Approximately 0.5 g of leaf tissue were groundwith a Polytron in 2 ml of GUS extraction buffer (50 mM NaPO4 pH 7.0, 1mM EDTA, 0.1% Triton X-100, 10 mM B-mercaptoethanol, 1 mM p-nitropherylglucuronide, 100 ug/ml bovine serum albumin, 0.02% sodium azide)incubated at 30° C. for 6 h and the reaction stopped with the additionof 0.4 ml of 2.5M 2-amino-2-methyl-1, 3-propanediol. The amount ofp-nitrophenol produced was calculated by measuring the absorbance at 415nm. A stopped reaction containing an identical amount of leaf extractwas used as a blank. Relative enzyme activities in the extracts werecalculated and expressed as nanomoles of p-nitrophenol produced per mgof protein per minute. Ten plants were screened and they all displayed alarge reduction in GUS activity levels relative to those observed inTTR-48. Five plants (TTR-1 to TTR-5) were chosen for a detailed analysisof the effects of anti-sense RNA inhibition of GUS gene activity. Theretransformed tobacco plants TTR-1 to TTR-5 (FIG. 14, lanes 1-5) allshowed a considerable to complete (lane 4) reduction in GUS activityregardless of the developmental stage of the leaves examined. Comparisonof the highest level of GUS activity observed in the original GUS+ plantTTR-48 (FIG. 14, lane 6) with the highest level found in any of theplants retransformed with PAL 1302 (lanes 1-5) shows that the reductionin GUS activity was at least 90%. The levels of GUS activity found inthe control plant TTR-88 (FIG. 14, lane 7) were similar to those of theoriginal GUS+ plant (lane 6) indicating that the retransformation andregeneration process undergone by TTR-48 were not responsible for thedecline in GUS activity observed in TTR-1 to TTR-5. Western blotanalysis of total protein extracts obtained from young leaves wereperformed. For extraction of leaf proteins, approximately 100 mg oftissue were ground in a 1.5 ml Eppendorf tube containing 0.7 ml of GUSextraction buffer described above. An equal volume of SDS PAGE 2X sampleloading buffer (1.3 M Tris-Cl pH 6.8, 2% B-mercaptoethanol, 50%glycerol, 5 mM EDTA, 0.1% bromophenol blue) was added and the samplesincubated at room temperature for 15 min. The extract was centrifuged at12,000 rpm for 2 min and the supernatant was frozen at -80° C. untiluse. Proteins were resolved in 10% SDS PAGE gels and immediatelytransferred electrophoretically onto a nitrocellulose filter. The GUSprotein was then detected in the gels using the rabbitanti-B-glucuronidase antibody obtained from Clonetech Laboratories andan anti-rabbit lgG alkaline phosphatase conjugate kit (Promega Biotech,Madison Wis., USA) according to the manufacturers instructions. Equalamounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE), transferred tonitrocellulose and cross-related with antibodies specific to the GUSprotein. The amount of GUS protein detected on the Western blotcorrelated well with the GUS activity found in the leaves of all thetobacco plants examined whether these displayed high levels of activitysuch as TTR-48 or no discernible activity as in TTR-3 (FIG. 14, lane 3).The reduction in GUS activity in TTR-1 to TTR-5 can therefore bedirectly attributed to lower quantities of GUS enzyme within theseplants. Southern blot analyses were performed to confirm that the senseGUS gene was still present and intact in TTR-1 to TTR-5 and to verifythat the anti-sense GUS construct had correctly integrated in the DNA ofthese plants. It was found that the arrangement of the original senseGUS gene in TTR-48 was unaffected by the transformation with theanti-sense gene containing vector, and it was also further ascertainedthat amongst the plants selected for this, analysis, between 1-3 copiesof the anti-sense gene were inserted into the genome of the plant whichcontained the sense gene. It was also determined that a single insertedanti-sense gene could lead to a near total reduction in sense geneactivity. Northern blot analyses were conducted on total leaf RNA todetermine if the reduction in the amounts of GUS enzyme observed inTTR-1 to TTR-5 correlated with their levels of GUS mRNA. Total RNA wasprepared from tobacco leaves by grinding 0.5 g of tissue in 2 ml ofextraction buffer (6 M guanidine-HCl, 0.1 M NaAc pH 6.0, 1.0%b-mercaptoethanol) for 10-30 sec using a Polytron. The mixture was thencentrifuged at 5,000 rpm for 3 min, the supernatant layered on an equalvolume of 5.7 M CsCl in 0.1 M NaCl, 10 mM Tris-Cl pH 7.5, 1 mM EDTA andcentrifuged again at 35,000 rpm for 16 h at 15° C. The resulting RNApellet was resuspended in 0.1 M NaAc pH 6.0, 0.1% SDS and extracted withan equal volume of phenol-chloroform (50:50, v/v). The aqueous phase wasadjusted to 0.3 M NaAc and the RNA precipitated with 2 volumes ofethanol. Following centrifugation, the pellet was washed in 70% ethanoland resuspended in sterile distilled water. RNA samples were resolved inthe presence of methylmercuric hydroxide in 1.3% agarose gels andtransferred onto a nylon membrane. The membranes were probed with (³² P)UTP labelled sense or anti-sense GUS RNA transcripts. These transcriptswere made from pGEM-GUS, a plasmid obtained by inserting the Bam HI-SstI fragment of pBI 221.1 (which contains the entire GUS coding sequence)into the Bam HI-Sst I sites of pGEM-4Z. Probes were made by cuttingpGEM-GUS with Eco RI and then using T7 RNA polymerase to provide atranscript which can hybridize to anti-sense GUS RNA or by digestionwith Hind III and transcription with SP6 RNA polymerase giving ananti-sense transcript that can hybridize to sense GUS mRNA only. Aradiolabelled sense-specific GUS RNA probe demonstrated that the levelsof GUS mRNA found in TTR-1 to TTR-5 were considerably lower than thoseobserved in TTR-48, the original GUS+ plant. As predicted, untransformedtobacco did not posses the GUS transcript. The levels of sense mRNAcorrelate well with the amount of GUS protein and activity observed inthese plants. Northern blot analysis using an anti-sense-specific GUSRNA probe demonstrated the presence of anti-sense GUS transcript in theretransformed plants The reduced amounts of GUS protein and GUS activityobserved in TTR-1 to TTR-5 can thus be attributed to the low levels ofGUS mRNA found in these plants. Low levels of GUS mRNA were alwaysassociated with the presence of the anti-sense GUS RNA. These resultsclearly indicate that a target sense gene can be successfully inhibitedusing anti-sense RNA.

EXAMPLE 1A

This example describes a method for isolating microspores in order toobtain genes that are essential to pollen formation and/or function.Microspores may be conveniently isolated by manual dissection of buds toremove anthers that are subsequently disrupted by gentle grinding in amortar and pestle in 10% sucrose. The extract is then filtered through a44 um nylon mesh and the microspores collected by centrifugation at 3000x g for one minute. The pelleted microspores are re-suspended in 10%sucrose, filtered and pelleted as before. Other methods of isolatingmicrospores can also be used.

Tissues other than microspores can be disrupted by a variety of methodsand the disrupted tissue used for RNA isolation. It is convenient todisrupt the tissue by using a motor driven homogenizer with 10 mls of asolution of 6M guanidinium HCl, 0.1 M Na acetate, pH 6.0, 0.1 Mbeta-mercaptoethanol per gram of tissue. The homogenate is cleared bycentrifugation at 5,000 x g and the supernatant is layered over asolution of 6M CsCl in Tris-EDTA buffer (TE buffer). Centrifugation at100,000 x g for 12-20 hrs at 15° C. is used to pellet RNA which issubsequently re-suspended in water and re-precipitated in the presenceof 0.3 M Na acetate and two volumes of ethanol. RNA is recovered bycentrifugation and re-suspended in water. The RNA obtained by suchmethod can be fractionated by oligo-dT cellulose chromatography toseparate the polyadenylated mRNA from the bulk of the non-polyadenylatedRNA. The microspore RNA is isolated by using a tight fitting motordriven glass homogenizer to disrupt the microspores. The homogenizationof 300 ul of packed microspores is conducted in 1 ml 6M guanidinium-HCl,0.1 M Na acetate, pH 6.0, 0.1 M beta-mercaptoethanol. The homogenate iscentrifuged at 5000 x g and the cleared supernatant is layered over asolution of 6M CsCl in TE buffer. An overnight centrifugation at 100,00x g is used to pellet the RNA which is subsequently re-suspended inwater and re-precipitated in the presence of 0.3 M Na acetate and twovolumes of ethanol. Other methods of RNA extraction can be used toobtain the RNA from the tissues described. Standard methodology usingoligo-dT cellulose is used to obtain polyadenylated mRNA from thesetotal RNA preparations.

EXAMPLE 2

This example relates to the use of constitutive promoters to regulatethe expression of pollen specific anti-sense RNA. The CaMV 35S promoterfrom pBl 221 was isolated as a Hind III-Xba I fragment and cloned in PAL1001 previously cut with Hind III and Xba I. This produced a vector thathad the CaMV 35S promoter joined to the nos ter and in between thepromoter and terminator were unique sites for: Xba I Bam HI, Sma I, KpnI and Sst I. This vector was named PAL 1007. PAL 1007 was digested withBam HI and to this digested vector was added a 2.4 Kb Bam HI fragmentcontaining a coding region from clone L16 in the anti-sense orientation.This vector was called PAL 1305 and was used to transform Brassicanapus. Transformation was carried out either using the method describedin Moloney, M. M., et al. (Plans Cell Reports (1989) 8:238-242) or,transformation can be carried out with surface sterilized stem epidermallayers. For this procedure, seeds of B. napus L. ssp. oleifera cv.Westar were sown in `Promix` mixed with 2 g/L of the slow-releasefertilizer `Nutricoate` in 8" pots. Plants were grown in the greenhouseunder a 16 photoperiod (using natural and artificial lighting). Forcoculture and regeneration experiments stem-sections from the top threestem internodes of approximately 1.5 month old plants were used (i.e.those with elongated floral spikes and several open flowers). Intactstem-sections were surface sterilized for 30 seconds in 70% ethanol and10 minutes in 1% sodium hypochlorite followed by three rinses in steriledistilled water. For transformation Agrobacterium tumefaciens GV 3101carrying the Ti plasmid pMP 90 to provide vir functions in trans and thebinary vector PAL 1110 was grown on YEP media (which consists of 10 gmper L of Yeast Extract, 10 gm per L of Bacto-pepetone and 5 gm per L ofNaCl, pH 7.0 containing 100 ugs per mL kanamycin for selection ofbacterial cells that contained the binary vectors). Cells were grownfrom one to two says at 28 C. The cells were collected by centrifugationand were resuspended at an approximate density of 10⁶ -10⁷ cells per mLin liquid EL which consists of MS micro- and macro- nutrients and B5vitamins containing 40 mg/L of FeNa-EDTA (obtained from BDH chemicals)and 3% sucrose, 10 mg/L BenzylAdenine, and 0.5 mg/L alpha naphthaleneacetic acid (NAA) and 18.8 mM KNO³ plus 20.6 mM NH₄ NO³. Medium wassolidified with 0.8% agar (Sigma) when the EL media was used for solidmedia plates.

The cell suspension was poured into the bottom of a sterile petri dishand sterilized stems were dissected directly in the bacterialsuspensions. The segments were sectioned longitudinally into halfsegments and cut into approximately 5 mm sections. The dissectedsegments were placed on filter paper discs on solid EL media for a 3 daycoculture under continuous fluorescent light (60 microeinsteins/m2/sec2)at 25 C. After a 2-3 day coculture, explants were transferred to solidEL media containing 500 ug/mL carbenicillin, and 100 ug/mL bekanamycin(Sigma). Shoots formed in 4-8 weeks, sections were transferred to freshsolid EL media with carbinicillin and bekanamycin every 3-4 weeks.Shoots that formed and did not bleach were excised and rooted on PDRmedia (B5-with 2% sucrose and 0.5 mg/L each of NAA and IAA). In somecases, green non-regenerating callus growing on selective medium wasseparated from explants and transferred to fresh medium to stimulateregeneration. Transformed plants were placed in a misting chamber, andafter two to four weeks transferred to the greenhouse. Plants were grownunder a 16 hour photoperiod and allowed to flower. Clonal propagationwas used to increase plant lines as well as hand crossing and selectionof seedlings from crossed plants on kanamycin containing media. Thismedia consisted of 0.8% agar, one-tenth MS salts an 100 ugs per mLbekanamycin with no sucrose in the media. Surface sterilized seeds wereused. The seeds were surface sterilized by rinsing in 70% ethanol for afew seconds, soaking in 1% sodium hypochlorite for 15 minutes, followedby rinsing three times in sterile distilled water. Seeds were place onthe surface of the agar in sterile dishes and allowed to sprout. Plantswhich did not carry the kanamycin gene linked to the anti-sense genebleached and died, while those that carried the antisense gene stayedgreen and were subsequently transferred to soil and allowed to flower.

EXAMPLE 3

In this example, another pollen specific coding region was used with thevector PAL 1007. In this case, a 1.3 Kb Hind III fragment from clone L19was isolated, made blunt ended and cloned into the Sma I site of pGEM4Z. This subclone was called pPAL 1914 and was then digested with Xba Iand Sst I. This fragment was added to Xba I-Sst I cut PAL 1007, givingrise to a vector called PAL 1307. This vector contains the CaMV 35Spromoter fused to a coding region from clone L19 in the anti-senseorientation. This vector was used to transform Brassica napus asdetailed in example 34.

EXAMPLE 4

This example relates to the use of inducible promoters to regulateexpression of pollen specific anti-sense RNA. In this example, the 1.2Kb Hind III-Pst I fragment of the D. melanogaster 70 KD heat shockprotein promoter was isolated from the subclone pPW 229 (Holmgren, R. etal., 1979, Cell 18:1359-1370) and cloned into Hind III-Pst I cut pGEM4Z. The heat shock promoter was excised as a Hind III-Sma I fragment andcloned into Hind III-Sma I cut PAL 1001. This produced a vector (PAL1009) that contains a heat shock promoter followed by a portion of thepolylinker and nos ter. The Sma I site was used to clone the 1.3 Kb HindIII fragment from clone L19 following making this fragment blunt ended.The clone containing this fragment in the anti-sense orientationrelative to the heat shock promoter was called PAL 1403. This vector wasused to transform Brassica napus as in example 34. Additionally, since asingle Eco RI site exists at the end of the nos ter in this construct, aselection marker gene was added to this construct, the enzymeBeta-glucuronidase driven by the CaMV 35S promoter using this unique EcoRI site for the insertion of this gene. This vector, which is the sameas PAL 1403 except that it now contains a convenient gene for scoringtransformation was named PAL 1408 and was used for transformation ofBrassica napus as described in example 34.

EXAMPLE 5

This example relates to the use of a pollen specific promoter tospecifically express pollen specific anti-sense RNA in pollen cells. Forthis, the vector PAL 1107 was used for the production of anti-sense RNAfrom the cDNA clone 4F described in FIG. 4. To construct this anti-sensevector, the Eco RI fragment from the cDNA clone 4F was isolated, madeblunt ended with Klenow and cloned into the Sma I site of PAL 1107. Avector that contained the cDNA clone in the anti-sense orientation (asdetermined by restriction enzyme analysis) was chosen. This vector wasnamed PAL 11074F and used to transform Brassica napus as described inexample 34.

EXAMPLE 6

For this example, the vector PAL 1107 was used for the production ofanti-sense RNA from the 2.4 Kb Bam HI fragment of clone L16 described inexample 2. To construct this anti-sense vector, the 2.4 Kb Bam HIfragment from clone L16 was isolated and cloned into the Bam HI site ofPAL 1107. A vector that contained this fragment in the anti-senseorientation (as determine by restriction enzyme analysis) was chosen.This vector was named PAL 1107-16CRAS and used to transform Brassicanapus as described in example 34.

EXAMPLE 7

For this example, the vector PAL 1107 was used for the production ofanti-sense RNA from the 1.3 Kb Hind III fragment of clone L19 describedin example 3. To construct this anti-sense vector, the 1.3 Kb Hind IIIfragment from clone L19 was isolated and made blunt ended and clonedinto the Sma I site of PAL 1107. A vector that contained this fragmentin the anti-sense orientation (as determine by restriction enzymeanalysis) was chosen. This vector was named PAL 1107-19CRAS and used totransform Brassica napus as described in example 34.

EXAMPLE 8

For this, the vector PAL 1107 was used for the production of anti-senseRNA from the cDNA clone related to clone L10 described in FIG. 5. Toconstruct this anti-sense vector, the Eco RI fragment from the cDNAclone was isolated, made blunt ended with Klenow and cloned into the SmaI site of PAL 1107. A vector that contained the cDNA clone in theante-sense orientation (as determined by restriction enzyme analysis)was chosen. This vector was named PAL 110710G and used to transformBrassica napus as described in example 34.

EXAMPLE 9

For this, the vector PAL 1107 was used for the production of anti-senseRNA from the cDNA clone related to clone L19 described in FIG. 6. Toconstruct this anti-sense vector, the Eco RI fragment from the cDNAclone was isolated, made blunt ended with Klenow and cloned into the SmaI site of PAL 1107. A vector that contained the cDNA clone in theanti-sense orientation (as determine by restriction enzyme analysis) waschosen. This vector was named PAL 110719 and used to transform Brassicanapus as described in example 34.

EXAMPLE 10

In this example, the pollen specific promoter in the vector PAL 1421 wasused for the production of anti-sense RNA using the cDNA clonehomologous to the gene contained with L10. This was accomplished byexcising the cDNA clone from the cloning vector with Eco RI and makingthis fragment blunt ended with Klenow. This blunt ended cDNA fragmentwas cloned into the Sma I site of PAL 1421. Clones were recovered thatcontained the cDNA insert in both orientations, and one was chosen thatcontained the insert in the anti-sense orientation relative to thepromoter of PAL 1421, resulting in the formation of a binarytransformation vector PAL 1492. PAL 1492 was used to transform Brassicanapus as described in example 34.

EXAMPLE 11

In this example, the pollen specific promoter in the vector PAL 1121 wasused for the production of anti-sense RNA using the cDNA clonehomologous to the gene contained within L10. This was accomplished byexcising the cDNA clone from the cloning vector with Eco RI and makingthis fragment blunt ended with Klenow. This blunt ended cDNA fragmentwas cloned into the Sma I site of PAL 1121. Clones were recovered thatcontained the cDNA insert in both orientations, and one was chosen thatcontained the insert in the anti-sense orientation relative to thepromoter of PAL 1121, resulting in the formation of a binarytransformation vector PAL 1110. PAL 1110 was used to transform Brassicanapus as described in example 34.

EXAMPLE 12

In this example, the pollen specific promoter from clone 19 was used forthe expression of pollen specific anti-sense RNA. The transformationvector PAL 1920 was digested with Sma I. To the Sma I site was added theDNA fragment corresponding to the cDNA clone homologous to clone L10 bydigesting the vector containing this clone with Eco RI and making thefragment blunt ended. This blunt ended cDNA fragment was cloned into theSma I site of PAL 1920. Clones were recovered that contained the cDNAinsert in both orientations, and one was chosen that contained theinsert in the anti-sense orientation relative to the promoter of pPAL1920, resulting in the formation of a binary transformation vector PAL1921. PAL 1921 was used to transform Brassica napus as described inexample 34.

EXAMPLE 13

In this example, anti-sense RNA is made specifically to the intronregion of clone L19 and a specific restorer gene is made which lacks theintron from clone L19. The construction of these two vectors is outlinedin FIG. 12. PAL 1954 was used to transform Brassica napus as in example34 to make a male sterile line. To create a specific restorer plantline, the vector PAL 1955 was used to transform Brassica napus as inexample 34.

EXAMPLE 14

In this example, the highly active promoter fragment of clone HP 101 weused to synthesize anti-sense RNA to the intron region of clone 19. Forthis example, the subclone containing the intron region of clone 19 thatis detailed in FIG. 12, pPAL 1914 was digested with Hind III and Bam HI.The promoter fragment from clone pPAL HP101 was added to this constructas a Hind III-Bam HI fragment giving rise to clones containing thepromoter fragment in the anti-sense orientation relative to the intron.This clone contained the promoter in an orientation such thattranscription of the promoter would cause the production of an RNA aportion of which would contain anti-sense RNA homologous to the intronregion of clone L19. This clone was called pPAL 19HP. The clone pPAL19HP was digested with Hind III and Sst I, and cloned into the vectorPAL 1001 using the Hind III and Sst I sites of PAL 1001, creating PALHP19. PAL HP19 was used to transform Brassica napus as in example 34. Itshould be noted that the vector PAL 1955 (see example 13 and FIG. 12)can be used for fertility restoration in plants that carry PAL HP19.

EXAMPLE 15

The pollen specific promoter contained in the subclone pPAL 0420 wasused for the construction of an anti-sense RNA gene under the control ofa pollen specific promoter as follows: A DNA fragment coding for apolyubiquitin of Arabadopsis was isolated from a plasmid which containsa polyubiquitin gene that has 5 copies of the ubiquitin monomericprotein was obtained from the University of Wisconsin, Madison, Wis.,USA and is described in Burke et al., Molecular and General Genetics, inpress. A Bam HI-Bgl II fragment was isolated that contains 3 of the 5copies of the polyubiquitin gene and this fragment was inserted intopPAL 0420 by using the single Bam HI site of the polylinker of PAL 0420.This gave rise to a plasmid containing the pollen specific promoter fromclone L4 followed by an DNA fragment containing ubiquitin codingsequences in the antisense orientation followed by the nos terpolyadenylation signal. This promoter/anti-sense gene construct wasexcised from pPAL 0420 by digestion with Eco RI. The Eco RI fragmentthat contains the promoter anti-sense gene was inserted into the Eco RIsite of the polylinker of BIN 19, resulting in the formation of a binarytransformation vector was named PAL 1479. PAL 1479 was used to transformBrassica napus as described in example 34.

EXAMPLE 16

The vector PAL 1479 was used so transform tobacco as described inexample 34.

EXAMPLE 17

The pollen specific promoter contained in the subclone pPAL 0420 wasused for the construction of a pollen specific anti-sense RNA gene asfollows: A DNA fragment coding for pine actin was obtained from J.Kenny-Byrne, Petawawa Canada. Two clones were obtained, Pac 1-A and Pac2, the clone Pac 1-A being described in: Canadian Journal of ForestryResearch (1988) 18:1592-1602, and the second clone Pac 2 (the sequencebeing closely homologous to that in Pac 1-A and the nucleotide sequenceof which having been submitted for publication). A Sph I fragment wasisolated from Pac 2 that contains the complete coding sequence of pineactin. This fragment also contains a small amount of 5' and 3'non-coding region. This Sph I fragment was cloned into the unique Sph Isite of pGEM 4Z. From this subclone, a Xba I fragment was isolated thatcontains only coding region and this Xba I fragment was cloned into theunique Eco RI site of pGEM 4Z. A clone was chosen that had theorientation such that the 5' end of the gene was next to the Bam HI sitein the polylinker and 3' end of the gene was next to the Sal I site inthe polylinker. This plasmid was called pPAL PAC. The actin codingregion was isolated form pPAL PAC by digestion with Bam HI and Sal I.This Bam HI-Sal I fragment was cloned into pPAL 0420 using the Bam HIand Sal I sites contained within the polylinker of pPAL 0420. This gaverise to a plasmid containing the pollen specific promoter from clone L4followed by an DNA fragment containing the actin coding sequence in theanti-sense orientation followed by the nos ter polyadenylation signal.This promoter/anti-sense gene construct was excised from pPAL 0420 bydigestion with Eco RI. The Eco RI fragment that contains the promoterantisense gene was inserted into the Eco RI site of the polylinker ofBIN 19, resulting in the formation of a binary transformation vector PAL1498. PAL 1498 was used to transform Brassica napus as described inexample 34.

EXAMPLE 18

The vector PAL 1498 was used to transform tobacco plants as outlined inexample 34.

EXAMPLE 19

In this example, tobacco that has been previously transformed tohygromycin resistance was transformed with an anti-sense gene thatspecifically blocks the hygromycin resistance in the pollen cells byvirtue of the fact that the anti-sense gene is under the control of apollen specific promoter in the vector PAL 1106. Transformed tobaccothat were resistant to hygromycin were obtained using the vector PAL1302 which is described in example 1. Selection of tobacco plant cellsresistant to hygromycin was via coculture with PAL 1302 and selection of50 ug per ml hygromycin. Southern blot analysis demonstrated thepresence of 5-6 copies of the sense hygromycin phosphotransferase genein one plant. This plant, referred to as TTR-122, was retransformed witha vector called PAL 1107A. Pal 1107A is the vector PAL 1106 to which hasbeen added the 0.8 Kb Bam HI hygromycin phosphotransferase fragmentisolated from PAL 1302 and inserted into PAL 1106 in the anti-senseorientation relative to the pollen specific promoter in PAL 1106. Theresulting vector was called PAL 1107A. Plants obtained from thistransformation were resistant to both hygromycin and kanamycin in theleaf tissue and were shown by southern blot analysis to contain theanti-sense gene. These plants were allowed to grow in the greenhouse andself fertilize. Clonal propagation of these plants were used as apreliminary increase of single plants, and these clonally propagatedplants were used for the production of male sterile plant line. Forexample, a plant which contained a single copy of the anti-sense geneand was derived from the transformation of TTR-122 was planted in a sandsoil mixture and allowed to grow in the greenhouse. This plant isreferred to as TTR-203. Measurement of the hygromycin phosphotransferaseactivity in this plant demonstrated high activity in leaves, petals,stigma and pistal and anther walls, but very low levels in pollen.TTR-122 showed high levels of hygromycin phosphotransferase activity inleaves, petals, stigma and pistal and anther walls and in pollen. Thisdemonstrated that the anti-sense gene was effective in blocking theexpression of the sense gene only in the pollen. Northern blot analysisconfirmed the presence of the anti-sense transcript specifically in thepollen of TTR-203 and also demonstrated low levels of the sense genemRNA. When flower buds first appeared, TTR-203 was watered three timesweekly with a solution of hygromycin (250 ugs per mL), thoroughlysaturating the sand soil mixture. This watering was continued forapproximately four weeks, or the period of time in which the majorflowering was occurring. Flowers produced during this time on plantscontaining the anti-sense gene were male sterile. Anther and pollenformation was inhibited, and mature pollen failed to develop. Femalefertility was unaffected by this treatment as hand pollination could beused for the pollination of the female portion of the male sterileflowers. Pollen that was from a hygromycin resistant plant was used forthe hybrid seed production. Watering of the plants with hygromycin wasstopped, and normal watering was resumed. Flowers that formed on plantsafter the hygromycin watering was stopped were male fertile and setselfed seed.

EXAMPLE 20

The pollen specific vector PAL 1107 was used for the production of malesterile plants The plant TTR-122 described above, was retransformed witha vector called PAL 1107HYGAS. Transformation was conducted as describedin example 34. Pal 1107HYGAS is the vector PAL 1107 to which has beenadded the 0.8 Kb Bam HI hygromycin phosphotransferase fragment isolatedfrom PAL 1302 and inserted into PAL 1107 in the anti-sense orientationrelative to the pollen specific promoter in PAL 1107. Plants obtainedfrom this transformation were resistant to both hygromycin and kanamycinin the leaf tissue and were shown by southern blot analysis to containthe anti-sense gene. These plants were allowed to grow in the greenhouseand self fertilize. Clonal propagation of these plants were used as apreliminary increase of single plants, and these clonally propagatedplants were used for the production of male sterile plant lines as inexample 19.

EXAMPLE 21

For the production of male sterile plants, the vector PAL 1419 was usedto transform Tobacco as outlined in example 34. The vector PAL 1419contains the pollen specific promoter from clone L4 controlling theexpression of the NPT II gene oriented in the anti-sense orientationrelative to the pollen specific promoter. This vector also contains aconstitutive version of the NPT II gene in the sense orientation drivenby the nos promoter. This vector therefore can confer resistance tokanamycin in all plant cells except pollen cells wherein the expressionof the sense gene is inhibited by the expression of the anti-sense genewhich is specifically expressed in the pollen. Tobacco plants wereobtained following transformation of the vector PAL 1419. These plantswere rooted and allowed to set flower. Plants were watered withkanamycin while flowering.

EXAMPLE 22

The vector PAL 1419 was used to transform Brassica napus as outlined inexample 34.

EXAMPLE 23

The vector PAL 1419 was used for the transformation of petunia leafdiscs.

EXAMPLE 24

In this example, the complete ricin A chain (pPAL Riccom) described inFIG. 13 was inserted into the vector PAL 1420 as a Bam HI fragment.Vectors were recovered that contained the ricin gene in both the senseand anti-sense orientation. A vector that contained the ricin A chain inthe sense orientation was recovered and called PAL 1420RIC. A vectorthat contained the gene in the anti-sense orientation was called PAL1420RICAS. PAL 1420RIC and PAL 1420RICAS were used for transformation ofBrassica napus as described in example 34 giving rise to plants thatcarried either the sense or anti-sense copy of the ricin A chain geneunder the control of the pollen specific promoter in PAL 1420.

EXAMPLE 25

In this example, the truncated version of the ricin A chain (pPAL Rictr)described in FIG. 13 was inserted into the vector PAL 1420 as a Bam HIfragment. Vectors were recovered that contained the truncated ricin genein both the sense and anti-sense orientation. A vector that containedthe ricin A chain in the sense orientation was recovered and called PAL1420tRIC. A vector that contained the gene in the anti-sense orientationwas called PAL 1420tRICAS. PAL 1420tRIC and PAL 1420tRICAS were used fortransformation of Brassica napus as in example 34 giving rise to plantsthat carried either the sense or anti-sense copy of the truncated ricinA chain gene under the control of the pollen specific promoter in PAL1420.

EXAMPLE 26

PAL 1420RIC and PAL 1420RICAS were used for transformation of tobaccoplants as in example 34, giving rise to plants that carried either thesense or anti-sense copy of the ricin A chain gene under the control ofthe pollen specific promoter in PAL 1420.

EXAMPLE 27

The vectors PAL 1420tRIC and PAL 1420tRICAS were used for transformationof tobacco plants as in example 34 giving rise to plants that carriedeither the sense or anti-sense copy of the truncated ricin A chain geneunder the control of the pollen specific promoter in PAL 1420.

EXAMPLE 28

For this example, the vector PAL 1423 was used to express the polylysinegene contained in the subclone pPAL pLys. The coding region from pPALpLys was isolated by Bam HI digestion and cloned in the senseorientation into Bam HI cut PAL 1423, giving rise to PAL 1487. PAL 1487was used to transform tobacco as in example 34.

EXAMPLE 29

For this example, the vector PAL 1920 was used to express the polylysinegene contained in the subclone pPAL pLys. The coding region from pPALpLys was isolated by Bam HI digestion and cloned in the senseorientation into Bam HI cut PAL 1920, giving rise to PAL 1987. PAL 1987was used to transform tobacco as in example 34.

EXAMPLE 30

In this example, a pollen specific promoter was used to synthesize aprotein molecule that is destructive to cellular function anddevelopment, namely the protease trypsin. The cDNA sequence coding fortrypsin has been described by Stevenson et al., Nucl Acids Res, 1986,14:8307-8330. The cDNA clone pMPt9 was obtained and used for theproduction of a modified trypsin molecule in which the N-terminal aminoacid residues were removed to give a protein that consisted of solelythe active protease form of trypsin and differed from the mature form inthat there was a methionine residue at the N-terminal position of themature protein, replacing the Isoleuecine found at this position in theactive mature protein. This was accomplished by digesting the plasmidpMPt9 with Fok I and Pst I, and recovering a fragment that encompassesnucleotides 81 to 835, the nucleotides after 835 being the G:C tail usedfor cloning of the cDNA, and treating this fragment with Klenow, andcloning into blunt ended Sma I cut M13mp19RF, and isolating a singlestranded phage clone that was used for site specific metagenesis tochange the isoleucine codon at nucleotides 84-86 in the publishedsequence from ATT to ATG, introducing a initiation codon where theisoleucine codon was. The mutated gene recovered was excised with Sal Iand Sst I, and inserted into PAL 1421 and named PAL 1456. The vector PAL1456 was used to transform tobacco as in example 34. It should be notedthat a restorer gene can be made if a trypsin inhibitor is inserted inan analogous fashion using a clone L4 derived promoter and transformedinto a male parent line. The expression of the trypsin inhibitor in thehybrid will specifically block the activity of the trypsin enzyme. Anumber of cDNA and genomic DNA sequences can be found for soybean andother trypsin inhibitors, for example see: Jofuku, K. D. and Goldberg,R. B., The Plant Cell 1989, 1:1079-1093.

EXAMPLE 31

In this example, we use a pollen specific promoter to synthesize theenzyme IamH specifically in pollen cells. The enzyme has activity thatcan cause the production of NAA from NAM, the substance NAA functioningas a plant hormone that is substantially toxic to developing pollengrains, while the precursor NAM being relatively non-toxic. For thisexample, the IamH gene was inserted into the vector PAL 1423. The IamHgene was isolated from pPCV311 as described in FIG. 19 and cloned as aBam HI-Sst I fragment in the Bam HI-Sst I sites of PAL 1423, creatingPAL 1424. This vector has the IamH gene (T-DNA gene 2) under the controlof a pollen specific promoter from clone L4. PAL 1424 was used totransform Tobacco as outlined in example 34.

EXAMPLE 32

The vector PAL 1424 was used to transform Brassica napus as outlined inexample 34.

EXAMPLE 33

In this example, the vector PAL 1107 was used for the production oftissue-specific GUS (beta-glucuronidase) enzyme. The gene for thisenzyme is available from Clonetech Laboratories, Palo Alto, Calif., USA.The gene was inserted into PAL 1107 as a Bam HI-SstI fragment and wasused to transform tobacco as in example 34. Plants produced haddetectable GUS activity only in developing pollen cells, and not anyother tissue tested. It should be noted that application of non-toxicanalog of glucuronic acid to which has been conjugated a toxic moleculesuch as glyphosate could be applied to these plants and cleavage of thetoxic moiety from the glucuronic acid would occur only in pollen cells.This provides an example of an enzyme that could be used for theproduction, in a tissue-specific fashion, of a toxic substance from anon-toxic analog.

EXAMPLE 34

This example describes methods used to transform tabacco and Brassicanapus.

For tobacco transformation, the tobacco cultivar, N. tabaccum, cv.Delgold was used. To accomplish this transformation, tobacco leaves lessthan 8 inches in length were surface sterilized by exposure to ethanolfor 5-6 seconds, then subsequent exposure to 1% sodium hypochlorite fora few minutes, usually 5-10 minutes, or until the cut edge of thepetiole turned white, then were rinsed several times in steriledistilled water. Leaf segments of approximately 0.5 to 1.0 squarecentimeters were excised from the sterile leaves, and were cocultured onshoot inducing media for two days with Agrobacterium tumefaciens GV 3101carrying the Ti plasmid pMP 90 to provide vir functions in trans(described by Koncz, C. and Schell, J., 1986, Mol. Gen. Genet.204:383-396) carrying the binary vector of interest. The vector isusually a derivative of Bin 19 which contains the NPT II gene driven bythe nopaline synthase promoter and terminated by the nos ter forselection of plant cells with kanamycin. Bin 19 is available fromClonetech Laboratories, Palo Alto, Calif., U.S.A. Transformed tobaccocells are selected on a shoot-inducing medium containing 0.8% agar, MSsalts, B5 vitamins, 3% sucrose, 1 mg per L of benzyladenine, 0.1 mg perL of alpha naphthalene acetic acid, (NAA) 300 μg/ml kanamycin and 500μg/ml carbenicillin (essentially as described by Horsch et al. 1985,Science, 227:1229-1231). Regenerated shoots are then transferred to aroot-inducing medium consisting of B5 medium with 2% sucrose, 500 μg/mlcarbenicillin and 0.5 mg/L each of NAA and indoleacetic acid (IAA).Rooted transformants are transferred to a misting chamber containinghigh humidity, after which the humidity is gradually lowered and plantsare subsequently transferred to the greenhouse.

For transformation of Brassica napus, the binary vector containingAgrobacterium strain GV 3101 carrying pMP 90 to provide vir functions intrans is used. Transformation was carried out either using the methoddescribed in Moloney, M. M., et al. (Plant Cell Reports (1989)8:238-242) or, transformation can be carried out with surface sterilizedstem epidermal layers. For this procedure, seeds of B. napus L. ssp.oleifera cv. Westar were sown in `Promix` mixed with 2 g/l of theslow-release fertilizer `Nutricoate` in 8" pots. Plants were grown inthe greenhouse under a 16 photoperiod (using natural and artificiallighting). For coculture and regeneration experiments stem-sections fromthe top three stem internodes of approximately 1.5 month old plants wereused (i.e. those with elongated floral spikes and several open flowers). Intact stem-sections were surface sterilized for 30 seconds in 70%ethanol and 10 minutes in 1% sodium hypochlorite followed by threerinses in sterile distilled water. For transformation Agrobacteriumtumefaciens GV 3101 carrying the Ti plasmid pMP 90 to provide virfunctions in trans and the binary vector of choice was grown on YEPmedia (which consists of 10 gm per L of Yeast Extract, 10 gm per L ofBacto-pepetone and 5 gm per L of NaCl, pH 7.0 containing 100 ugs per mLkanamycin for selection of bacterial cells that contained the binaryvectors). Cells were grown from one to two days at 28 C. The cells werecollected by centrifugation and were resuspended at an approximatedensity of 10⁶ -10⁷ cells per mL in liquid EL which consists of MSmicro- and macro-nutrients and B5 vitamins containing 40 mg/L ofFeNa-EDTA (obtained from BDH chemicals) and 3% sucrose, 10 mg/LBenzylAdenine, and 0.5 mg/L alpha naphthalene acetic acid (NAA) and 18.8mM KNO³ plus 20.6 mM NH₄ NO³. Medium was solidified with 0.8% agar(Sigma) when the EL media was used for solid media plates.

The cell suspension was poured into the bottom of a sterile petri dishand sterilized stems were dissected directly in the bacterialsuspensions. The segments were sectioned longitudinally into halfsegments and cut into approximately 5 mm sections. The dissectedsegments were placed on filter paper disc on solid EL media for a 3 daycoculture under continuous fluorescent light (60 microeinsteins/m2/sec2)at 25 C. After a 2-3 day coculture, explants were transferred to solidEL media containing 500 ug/mL carbenicillin, and 100 ug/mL bekanamycin(Sigma). Shoots formed in 4-8 weeks, sections were transferred to freshsolid EL media with carbinicillin and bekanamycin every 3-4 weeks.Shoots that formed and did not bleach were excised and rooted on PDRmedia (B5- with 2% sucrose and 0.5 mg/L each of NAA and IAA). In somecases, green non-regenerating callus growing on selective medium wasseparated from explants and transferred to fresh medium to stimulateregeneration. Transformed plants were placed in misting chamber, andafter two-four weeks transferred to the greenhouse. Plants were grownunder a 16 hour photoperiod and allowed to flower.

Clonal propagation was used to increase plant lines as well as handcrossing and selection of seedlings from crossed plants on kanamycincontaining media. This media consisted of 0.8% agar, one-tenth MS saltsand 100 ugs per mL bekanamycin (available from Sigma Chemicals, St.Louis, Mo., U.S.A.) with no sucrose in the media. Surface sterilizedseeds were used. The seeds were surface sterilized by rinsing in 70%ethanol for a few seconds, soaking in 1% sodium hypochlorate for 15minutes, followed by rinsing three times in sterile distilled water.Seeds were placed on the surface of the agar in sterile dishes andallowed to sprout. Plants which did not carry the kanamycin gene linkedto the antisense gene bleached and died, while those that carried theantisense gene stayed green and were subsequently transferred to soiland allowed to flower.

EXAMPLE 35

This example describes the isolation of two genes involved in tumorformation in plant tissues following infection with Agrobacterium, theIamS and the IamH genes from the Ti plasmid of the Agrobacteriumtumefaciens strain C58. The isolation of the IamH gene is particularlydescribed. The source of DNA coding for these genes was the plasmid pPCV311. The plasmid pPCV311 is described in: Koncz, C. and Schell, J.,Molecular and General Genetics, (1986), 204:383-396, and contains theoncogenic region of the T-DNA plasmid contained in the C58 strain ofAgrobacterium. The plasmid pPCV 311, contains a region of T-DNA thatwhen transferred to plant cells causes tumor formation. This oncogenicregion of the T-DNA is entirely contained in the plasmid pPCV-311. Thisregion of DNA contains four genes, that when expressed in plant cellsare sufficient for tumor formation. The approximate ceding regions ofthese four genes and the direction of transcription of these four genesare indicated in FIG. 15. The other portions of the vector pPCV 311 arenot shown in that they are not relative to the following constructions.Additionally, the oncogenic region of the Agrobacterium strain C58 islocated on the T-DNA plasmid within that bacterium, commonly referred toas the wild-type nopaline plasmid. A nearly identical oncogenic regionis also found in wild type octopine strains which could also be used asa source of genes. The complete nucleotide sequence of an octopinestrain oncogenic region is described by Barker et al., Plant MolecularBiology 2:335-350 (1983). The partial sequence obtained from variousconstructs of genes derived from pPCV 311 was compared to the publishednucleotide sequence.

Two genes were isolated from pPCV 311, the lamH and the lamS genes,commonly referred to as genes 2 and 1 respectively. The lamH gene wasisolated by first subcloning the indicated Hind III fragment, a fragmentthat contains all of the coding region of gene 2 and additional 5'sequences that were subsequently removed for the construction of apromoterless version of the gene. The restriction sites mapped in thissubclone are shown in FIG. 15 and the subclone is referred to as pPALG2. For the isolation of coding sequences only, pPAL G2 was first splitinto two smaller clones and the gene later reconstructed. The Xba I-SmaI and Sma I-Sma I fragments shown in FIG. 15 were isolated by gelelution and subsequently cloned into the following vectors: The SmaI-Sma I fragment was cloned into pGEM 4Z, giving rise to pPAL 899. TheXba I-Sma I fragment was subcloned into pGEM 7Z, giving rise to pPAL898. The 5' non-coding sequences of the lamH gene that are present inthis subclone were removed in the following fashion: pPAL 898 wasdigested with Eco RI, the Eco RI site is in the promoter region of theclone, and in this subclone is the only Eco RI site. This digested DNAwas then treated with Exonuclease III, and following digestion treatedwith S1 nuclease and the Klenow fragment of DNA polymerase I. Thetreated DNA was then cut with Pst I and treated with Klenow fragment inorder to make the Pst I site blunt. The linear, digested, blunt endedplasmid was then relegated and used to transform E. coli DH5-alphaaccording to standard protocols. Subclones were chosen, sequenced andone subclone was chosen that was deleted to 8 nucleotides in front ofthe ATG start of translation codon. The ATG start codon was determinedby comparison of the nucleotide sequence obtained from the deletedsubclones to the nucleotide sequence for the octopine strain describedby Barker, et al. Plant Molecular Biology 2:335-350 (1983). Thenucleotide sequences of both the 5' non-coding and the coding regionwere nearly identical. This subclone was named pPAL 897, the ATG codonis shown in FIG. 15, the direction of transcription in this case wouldbe from right to left in FIG. 15. The plasmid contained the 5' half ofthe coding region from the lamH gene, with the promoter sequencesdeleted.

The construction of the 3' half of the lamH gene, contained in theplasmid pPAL 898 was carried out as follows. A 3' region of the genethat contains the polyadenylation signal naturally found in the gene wasisolated by digestion pPAL 898 with the enzymes Bam HI and Apa I. Thedigested DNA was treated with Klenow fragment to make it blunt ended andwas relegated. This gave rise to the subclone pPAL 896, which is aplasmid that contains the 3' half of the lamH gene. To reconstruct theintact lamH gene, pPAL 896 was digested with Hind III and Sma I, and the3' half gene fragment was isolated by gel elution. pPAL 897 was digestedwith Sma I and Hind III and the isolated 3' fragment from pPAL 896 wascloned into these sites, reconstructing a promoterless version of thegene that contains the indicated array of restriction sites flanking thegene. This plasmid was named pPAL 895 and is shown in FIG. 15.

EXAMPLE 36

This example describes the isolation and construction of a promoterlessversion of the gene 1, IamS: indole acetamide synthase gene of the Tiplasmid of the Agrobacterium tumefaciens strain C58 which procedure issummarized in FIG. 16. The gene was isolated from the plasmid pPCV311.The Sma I-Pst I fragment that contains 5' and 3' regions of the lamSgene as well as the coding region was isolated by gel elution andsubcloned into a derivative of pGEM 4Z called pGEM-noEco. pGEM-noEco isa plasmid from which the Eco RI site of pGEM 4Z has been removed bycutting with Eco RI and making blunt ended and relegating such that onlythe Eco RI site was removed. The fragment was inserted in theorientation shown relative to the polylinker. This subclone was calledpPAL 889. pPAL 889 was digested with Eco RI, and briefly treated withExonuclease III, followed by S1 nuclease. The DNA was digested with SmaI and treated with Klenow fragment to make it blunt ended. The DNA wasrelegated and clones recovered. Some of these clones were chosen,sequenced, and one clone was found which had 5' sequences deleted suchthat only approximate 15 bases upstream of the ATG start of translationcodon remained. This plasmid was named pPAL 888. The Kpn I site at the5' end of the gene as well as the Pst I site at the 3' end of the genewere both converted to Sal I sites by cutting with Kpn I, end fillingwith Klenow and adding synthetic Sal I linkers, and repeating the linkeraddition at the Pst I site such that the entire gene can be excised as asingle Sal I fragment. This plasmid was named pPAL 887. This plasmidcontains the promoterless version of the lamS gene and contains thearray of restriction sites shown that flank the gene as shown in FIG.16.

EXAMPLE 37

This example relates to the detailed characterization of the codingregions of clone number L4 a microspore specific clone isolated from aBrassica napus genomic library and the construction of vectorscontaining these genes. FIG. 2a is a schematic representation of therestriction map and coding regions of clone number L4. The clonecontains three different members of the same gene family. These genesare identified as Bp4A, Bp4B and Bp4C. The first (Bp4A) and third (Bp4C)genes are functional, the second gene has modifications that most likelyrender it non-functional. The restriction map is made diagramatically inthat the non-transcribed regions are shown as a single line, while thetranscribed regions are shown as a boxed area. The second gene (Bp4B) isidentified on the basis of sequence homology and is therefore boxed witha dotted line. The notation "del 220" refers to an approximately 220base pair deletion/rearrangement affecting the second gene (Bp4B) inthis clone. Start of transcription is located at the leftmost side ofeach boxed area (except in the case of gene Bp4B) and exon and intronpositions are noted by the exons being filled in with black and theintron positions being left unfilled. A small arrowhead is shown on thenon-transcribed 5' region of each gene, this arrowhead serves toindicate the promoter region of each gene. Restriction sites areidentified such that the number of the first nucleotide of therestriction enzyme recognition site is shown. Not all restriction sitesare shown, only those relative to the constructs detailed within areindicated. The genes are presented with the 5' region being on the leftside and the 3' region being on the right. The numbering of the DNAsequence in all cases starts from left to right, 5' to 3'.

In FIG. 3a, the DNA sequence of the clone L4 is shown, the orientationof the sequence and the genes contained within the microspore specificclone from Brassica napus are from 5' to 3'. In FIG. 3a, clone L4,nucleotide 1 in the complete sequence is at the left-most Eco R1 sitewhile nucleotide 8579 is at the first nucleotide of the right-most EcoR1 site. The start of transcription of gene 1 in clone L4 is nucleotide235. The 5' and 3' intron splice sites are identified in boldface type.The start ATG codon is shown as well as the stop termination codon. Thededuced amino acid sequence of the proteins encoded for by these genesare also shown. The end of transcription for gene 1 is approximatelynucleotide 1427. As indicated above the second gene in clone L4 is mostlikely non-functional due to an insertion and a deletion that occurs inthe region of the promoter and first exon. This gene was not utilizedfor constructs. The third gene in clone L4 has a transcriptional startat position number 6298 in the DNA sequence and transcription ends atapproximately nucleotide 7490. The ATG start codon, intron splice sitesand termination stop codon are all identified as above. Vectors wereconstructed from this clone by using promoter fragments from both genes1 and 2. The specific promoter fragment constructs are detailed below.

The construction of 2 vectors containing promoter and promoter fragmentsfrom the clone L4 was carried out as follows and as shown in FIG. 7(A,B,C,D) The first vector was constructed by first subcloning the EcoR1-Sst 1 (nucl. 1-2132) fragment containing the first gene of clone L4(235 base pairs of promoter/exon/intron/second exon) in the commerciallyavailable vector pGEM-4Z (Promega Biotech, Madison, Wis., U.S.A.) usingthe Eco R1-Sst 1 sites of the polylinker of this vector. This plasmidwas named pPAL 0402. The 2.7 Kb Eco RI fragment of clone L4 thatcontains the third gene (Bp4C) was then cloned into the Eco RI site ofpGEM 4Z, leading to a plasmid called pPAL 0411. The plasmid pPAL 0402was then digested with Eco R1 and the 2.7 Kb Eco R1 fragment from pPAL0411 (nucl. 5859-8579) that contains the gene number three (Bp4C) fromclone L4 was added to it. Clones were recovered that contained thisinserted 2.7 Kb Eco R1 fragment in both orientations relative to thepromoter region of the first gene. A clone that contained this thirdgene fragment in an orientation such that the promoter from the thirdgene was opposite to the promoter in the first gene was chosen andcalled pPAL 0403. pPAL 0403 contains the entire third gene from clone L4oriented in such a fashion as to have the promoter region immediatelyadjacent to the 235 base pair promoter region of the first gene in pPAL0403. This plasmid, pPAL 0403 was digested with Dde 1. Dde 1 digestionproduced a fragment of approximately 1.9 Kb. The Dde I sites are locatedat nucleotides 303 and 7366. Because of the orientation of thesefragments, digestion with Dde I produces a 1.9 Kb fragment. This 1.9 Kbfragment contains a copy of the third gene (Bp4C) oriented such that thedirection of transcription of this third gene is from right to left,fused to the 235 base pair promoter fragment from the first gene ofclone L4 Bp4A) which is transcribed from left to right, ending in a DdeI site that is located 67 basepairs down stream of the major start siteof transcription and proceeds the ATG start of translation codon by 2nucleotides. This 1.9 Kb Dde I fragment made blunt with Klenow fragmentand cloned into the Xba I site of the polylinker region of pGEM 4Zpreviously made blunt ended with Klenow fragment. The resultant plasmidpPAL0408, was recovered and subsequently was digested with Sal I and SstI, which releases the cloned Dde I fragment bordered by on the left handside, (nucl 7366) Sal I and on the right hand side (nucl 303) of thisconstruct and contains a portion of the polylinker of pGEM 4Z containingthe following unique sites: Bam HI, Sma I, Kpn I, and Sst I restructionenzyme sites. This Sal I-Sst I fragment was cloned into the Sal I-Sst Isotes of PAL 1001. PAL 1001 is the binary vector Bin 19 (described byBevan, M., Nucleic Acids Res., 1984, 12:8711-8721) to which has beenadded the nor ter polyadenylation signal as a 260 bp Sst I-Eco RIfragment isolated from the plasmid pRAJ 221 (available from ClonetechLaboratories, Palo Alto, Calif., U.S.A.) in the Sst I-Eco RI sites ofthe polylinker region of Bin 19. The nos ter is identified as a stippledbox. The binary transformation vector that resulted from the insertionof the Sal I-Sst I fragment of pPAL 0408 into PAL 1001 was named PAL1107. The details of the construction are shown in FIG. 7A. This vectorhas a copy of the third gene oriented such that the direction oftranscription of this third gene is from right to left, fused to the 235base pair promoter fragment from the first gene of clone L4 which istranscribed from left to right, followed by a polylinker with uniquesites for the insertion of DNA which consist of: Bam HI, Sma I, Kpn Iand Sst I followed by the nos ter. This vector has the feature in thatadditional 5' non-coding sequences were placed upstream to the 235 basepair core promoter on Bp4A, but these additional 5' sequences were in anopposite orientation. The provision of these sequences in thisorientation does not affect the pollen specificity of the core 235 basepair promoter. With this vector, one can also ascertain for positioneffects of the transformation process in that since the vector containsan intact copy of the Bp4C gene, probing for the levels of expression ofthe Bp4C gene can give an indication as to what levels of expressionfrom the other pollen specific promoter in PAL 1107 can be expected.

EXAMPLE 38

This example describes the construction of additional vectors having thepromoter regions of the genes contained in clone L4 whcih may be usefulfor pollen specific expression of gene sequences. The three genes withinclone L4 (Bp4A, Bp4B, Bp4C) show very near-exact DNA homology and thisis most apparent between the first (Bp4A) and third (Bp4C) gene. Thesecond gene (Bp4B) is a homologous copy that has undergone sequencechanges that have appear to have lead to inactivation of it. Theextensive similarity between the first, second and third genes in cloneL4 is also maintained in the promoter region such that out of the first235 nucleotides of the first and third gene promoter regions there areonly 5 nucleotides that differ between them. Downstream of the TATA boxin these two promoters the only difference between them is the presenceof one additional nucleotide at the start of transcription (e.g.Promoter 1,Bp4A: . . . TATGTTTtAAAA . . . versus promoter 3,3p4c: . . .TATGTTTAAA (transcribed region underlined, single nucleotide differenceshown in lower case). However, within the sequence of the first genethere is a nucleotide change that introduces a Dde I site (nucl 303) inthe untranslated 5' leader sequence upstream of the ATG start codon thatis not present in the untranscribed leader sequence of the third gene inclone L4. For this reason, chimeric promoter constructs were made whichutilized this Dde I site in the first gene joined to sequences from thethird gene promoter. The region of the first promoter used for theseconstructs consisted of the sequences contained between the Sna BI site(nucl 210) near the TATA box to the Dde I site located immediatelyupstream of the ATG start codon in the first gene (nucleotide 303 is thefirst nucleotide in the recognition sequence for Dde I). The otherregion of this chimeric promoter (5' of the TATA box) was a fragmentextending from the Eco RI site of the third promoter (nucleotide 5858)to the Sna BI site near the TATA box (nucleotide 6273). Therefore tofacilitate construction of these pollen specific vectors, the followingreconstructions were performed.

The Eco RI to Dde I fragment that encompasses the promoter region of thefirst gene in clone L4 was isolated by first cutting pPAL 0402 with DdeI, blunting with Klenow, and then cutting with Eco RI. The 235 base pairfragment corresponding to this region was cloned into the Eco RI-Sma Isites of pGEM 4Z. This plasmid (pPAL 0422), was then cut with Eco RI andSna BI. A DNA fragment that contained the Eco RI to Sna BI portion ofthe promoter for gene 3 in clone L4 was isolated by digesting pPAL 0411with Eco RI and Sna BI. This released an approximately 415 base pair EcoRI (nucl 5858) to Sna BI (nucl 6273) fragment that represents most ofthe 5' region of the gene 3' promoter from clone L4 (the Sna BIrecognition site is 2 base pairs downstream of the TATA box). This EcoRI-Sna BI fragment was used to replace the shorter Eco RI-Sna BIfragment removed from the first promoter subclone (pPAL 0422),reconstructing a promoter fragment of approximately 550 base pairs. Thisplasmid is referred to as pPAL 0421. This chimeric promoter fragmentcontains 415 base pairs of the promoter of gene three in clone L4,followed by approximately 99 nucleotides of the first genepromoter/untranslated leader sequence.

For construction of a pollen specific cassette vector, the followingplasmids were first constructed. The first plasmid constructed containedthe nos ter polyadenylation signal with a polylinker in front of the noster. This was accomplished by first isolating from pRAJ 221 the nos teras a Sst I-Eco RI fragment and this fragment was cloned in pGEM 4Z usingthe Sst I and Eco RI sites in the polylinker. This subclone is referredto as pPAL 001. To pPAL 001, a fragment coding for neomycinphosphotransferase (NPT II) derived from the plasmid pRAJ 162 was addedto it in the antisense orientation as follows: The plasmid pRAJ 162contains the NPT II gene from the transposon TN 5 inserted as a Sal Ifragment and bounded by a polylinker in the plasmid pUC-9 and wasobtained from the Plant Breeding Institute, Cambridge, U.K. pRAJ 162 wasdigested with Hind III and Sma I. The DNA fragment containing the NPT IIgene was isolated by elution from an agarose gel. pPAL 001 was digestedwith Hind III and Sma I and the NPT II gene fragment was ligated intoit. The resultant plasmid was called pPAL 002 and had such orientationof restriction sites and the NPT II gene and nos ter as follows: HindIII, Pst I, Sal I, 3' end NPT II coding sequence 5' end, Sal I, Bam HI,Sma I, Kpn I, Sst I, nos ter, Eco RI. pPAL 002 was cut with Hind III andthe site made blunt ended by the use of Klenow fragment. pPAL 0421 wasdigested with Hinc II and Pvu II, both of which leave blunt ends, andthe promoter fragment was ligated into Hind III cut blunt ended pPAL002. Plasmids were obtained that contained the promoter in bothorientations relative to the nos ter. One plasmid was chosen with theproper orientation (5' promoter/antisense NPT II/nos ter) and was namedpPAL 0419. pPAL 0419 has the following DNA fragments: A small (approx.130 bp) of pGEM 4Z that contains the SP6 promoter, the 550 base pairchimearic promoter, the NPT II gene in the antisense orientationrelative to the promoter, followed by the nos ter polyadenylationsignal. This entire promoter/NPT lI/nos ter construct is excisable byEco RI. In order to provide promoter sequences that could be utlizedwith additional gene constructs, the plasmid pPAL 0419 was digested withSal I. This digest removes the NPT II coding region and this Sal Idigested pPAL 0419 was relegated giving rise to pPAL 0402. pPAL 0420represents the pollen specific promoter followed by a polylinker forinsertion of genes that has the following unique sites: Hinc II, Pst I,Sal I, Bam HI, Sma I, Kpn I, Sst I, followed by the nos terpolyadenylation signal. The entire promoter/polylinker/nos ter constructcan be conveniently excised as a single Eco RI fragment. The details ofthis construct is shown in FIG. 7B. This plasmid was used for theconstruction of an additional pollen specific promoter in a binarytransformation vector. The intact L4 clone in the lambda cloning vectorwas digested to completion with the restriction enzymes Sst I and Hha I.The resultant fragments were separated by gel electrophoresis and a 2.65Kb fragment that contains the promoter/first exon/intron/partial secondexon region of gene three in clone L4 and corresponds to nucleotides4565 to 7216 in the sequence of clone L4 was isolated. This fragment wasmade blunt ended with Klenow and cloned into the binary transformationvector PAL 1001 previously described with reference to FIG. 7a. PAL 1001was first cut with Hind III and made blunt ended with Klenow. Clonescontaining this fragment (promoter/first exon/intron/partial secondexon) were recovered. A clone was chosen that contained this fragment inthe proper orientation such that the direction of transcription wastowards the nos ter in PAL 1001. This vector was named PAL 1421. Thisvector contains approximately 1.9 kb of upstream promoter region fromthe gene 3 in clone L4 followed by the first exon, the complete intronand 15 bases of the second exon of gene three followed by a polylinkercontaining the following unique sites: Sal I, Xba I, Bam HI, Sma I, KpnI, Sst I, and finally the nos term polyadenylation signal. A varient ofthis vector was constructed by digesting PAL 1421 with Eco RI andreplacing the promoter/exon/intron/ second exon/polylinker/nos terstructure with the promoter/polylinker/nos ter structure from pPAL 0420using Eco RI such that a longer 5' promoter region is reconstructed inthe binary transformation vector. The resultant vector was named PAL1423. The outline of this construction is shown in FIG. 7D.

EXAMPLE 39

In this example, a pollen specific promoter is used to synthesize theenzyme IamH specifically in pollen cells. The enzyme has activity thatcan cause the production of NAA from NAM, the substance NAA functioningas a plant hormone that is substantially toxic to developing pollengrains, while the precursor NAM being relatively non-toxic. For thisexample, the lamH gene was inserted into the vector PAL 1423. The lamHgene was isolated from pPCV311 as described in FIG. 15 and cloned as aSal I fragment in the Sal I site of PAL 1423, creating PAL 1426. Thisvector has the IamH gene (T-DNA gene 2) under the control of a pollenspecific promoter from clone L4 in the sense orientation. PAL 1426 wasused to transform Tobacco as outlined in Example 34.

EXAMPLE 40

The vector PAL 1426 was used to transform Brassica napus as described inExample 34.

EXAMPLE 41

In this example, the vector PAL 1107 was used for the production oftissue-specific GUS beta-glucuronidase) enzyme. The gene for this enzymeis available from Clonetech Laboratories, Palo Alto. Calif., U.S.A. Thegene was inserted to PAL 1107 as a Bam HI-Sst I fragment and was used totransform tobacco as described in Example 34. Plants produced haddetectable GUS activity only in developing pollen cells, and not anyother tissue tested.

The application of a non-toxic analog of glucuronic acid to which hasbeen conjugated a toxic molecule could be applied to these plants andcleavage of the toxic moiety from the glucuronic acid would occur onlyin pollen cells. This provides an example of an enzyme that could beused for the production, in a tissue-specific fashion, of a toxicsubstance from a non-toxic analog. One such analog that could be used isGluc-Camp, a glucuronic acid analog that is conjugated tochloramphenicol. When acted upon by glucoronidase, chloramphenicol isproduced, inhibiting cellular growth and development.

EXAMPLE 42

In this example, two isogenic plant lines (A1, A2) were produced thatcarried either the IamS or the lamH genes. Tobacco plants weretransformed with PAL 1426 containing the IamH gene as in Example 39,producing the A2 line. The IamS gene described in FIG. 16 was insertedas a Sal I fragment into the vector PAL 1423 in the sense orientation,giving rise to PAL 1425. PAL 1425 was used to transform tobacco asdescribed and tobacco plants were produced that carried PAL 1425. Theselines represented the A1 lines. Tobacco plants that contained both PAL1426 and PAL 1425 were selfed and homozygous A1 and A2 lines wereselected.

EXAMPLE 43

In this example, PAL 1426 (see Example 39) and PAL 1425 were used totransform Brassica napus. Plants lines homozygous for the A1 and A2genes were selected as in Example 42.

We claim:
 1. An isolated recombinant DNA molecule comprising:(a) a DNAsequence which encodes a gene product which, when produced in a cell ofa plant, is capable of converting a non-toxic substance to a cytotoxicsubstance, and the cytotoxic substance is capable of interfering withthe function or development of said cell; and (b) a promoter capable ofcausing the transcription of said DNA sequence preferentially in cellscritical to pollen formation or function.
 2. A method of producinghybrid plant seed comprising crossing a genetically transformedmale-sterile plant with a male-fertile male parent plant,wherein saidgenetically transformed male-sterile plant comprises a first recombinantDNA molecule according to claim 1, and wherein said male-fertile maleparent plant comprises a second recombinant DNA sequence whichcompensates for the effects caused by expression of said firstrecombinant DNA molecule.
 3. A method for producing a male sterile plantcomprising the steps of:(a) obtaining a transformed plant cell that hasbeen genetically transformed with a DNA molecule according to claim 1;(b) regenerating from said transformed plant cell a geneticallytransformed plant; and growing the plant under conditions where malesterility is achieved.
 4. Hybrid seed of a pollen-producing plant havinga nuclear genome incorporating one or more recombinant DNA molecules asclaimed in claim 1, and at least one restorer DNA molecule which negatesthe effects caused by expression of said one or more recombinant DNAmolecules.
 5. A method of producing hybrid plant seed comprisingcrossing a genetically transformed male-sterile plant with amale-fertile male parent plant, wherein said genetically transformedmale-sterile plant comprises a first recombinant DNA molecule accordingto claim 1.